Cold-adapted equine influenza viruses

ABSTRACT

The present invention provides experimentally-generated cold-adapted equine influenza viruses, and reassortant influenza A viruses comprising at least one genome segment of such an equine influenza virus, wherein the equine influenza virus genome segment confers at least one identifying phenotype of the cold-adapted equine influenza virus, such as cold-adaptation, temperature sensitivity, dominant interference, or attenuation. Such viruses are formulated into therapeutic compositions to protect animals from diseases caused by influenza A viruses, and in particular, to protect horses from disease caused by equine influenza virus. The present invention also includes methods to protect animals from diseases caused by influenza A virus utilizing the claimed therapeutic compositions. Such methods include using a therapeutic composition as a vaccine to generate a protective immune response in an animal prior to exposure to a virulent virus, and using a therapeutic composition as a treatment for an animal that has been recently infected with a virulent virus, or is likely to be subsequently exposed to virulent virus in a few days whereby the therapeutic composition interferes with the growth of the virulent virus, even in the absence of immunity. The present invention also provides methods to produce cold-adapted equine influenza viruses, and reassortant influenza A viruses having at least one genome segment of an equine influenza virus generated by cold-adaptation.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a division of application Ser. No. 09/506,286 filed Feb. 16, 2000, now U.S. Pat. No. 6,482,414, which is a continuation in part of application Ser. No. 09/133,921 filed Aug. 13, 1998, now U.S. Pat. No. 6,177,082, and continuation in part of PCT/US99/18583 filed Aug. 12, 1999.

FIELD OF THE INVENTION

The present invention relates to experimentally-generated cold-adapted equine influenza viruses, and particularly to cold-adapted equine influenza viruses having additional phenotypes, such as attenuation, dominant interference, or temperature sensitivity. The invention also includes reassortant influenza A viruses which contain at least one genome segment from such an equine influenza virus, such that the reassortant virus includes certain phenotypes of the donor equine influenza virus. The invention further includes genetically-engineered equine influenza viruses, produced through reverse genetics, which comprise certain identifying phenotypes of a cold-adapted equine influenza virus of the present invention. The present invention also relates to the use of these viruses in therapeutic compositions to protect animals from diseases caused by influenza viruses.

BACKGROUND OF THE INVENTION

Equine influenza virus has been recognized as a major respiratory pathogen in horses since about 1956. Disease symptoms caused by equine influenza virus can be severe, and are often followed by secondary bacterial infections. Two subtypes of equine influenza virus are recognized, namely subtype-1, the prototype being A/Equine/Prague/1/56 (H7N7), and subtype-2, the prototype being A/Equine/Miami/1/63 (H3N8). Presently, the predominant virus subtype is subtype-2, which has further diverged among Eurasian and North American isolates in recent years.

The currently licensed vaccine for equine influenza is an inactivated (killed) virus vaccine. This vaccine provides minimal, if any, protection for horses, and can produce undesirable side effects, for example, inflammatory reactions at the site of injection. See, e.g., Mumford, 1987, Equine Infectious Disease IV, 207-217, and Mumford, et al., 1993, Vaccine 11, 1172-1174. Furthermore, current modalities cannot be used in young foals, because they cannot overcome maternal immunity, and can induce tolerance in a younger animal. Based on the severity of disease, there remains a need for safe, effective therapeutic compositions to protect horses against equine influenza disease.

Production of therapeutic compositions comprising cold-adapted human influenza viruses is described, for example, in Maassab, et al., 1960, Nature 7, 612-614, and Maassab, et al., 1969, J. Immunol. 102, 728-732. Furthermore, these researchers noted that cold-adapted human influenza viruses, i.e., viruses that have been adapted to grow at lower than normal temperatures, tend to have a phenotype wherein the virus is temperature sensitive; that is, the virus does not grow well at certain higher, non-permissive temperatures at which the wild-type virus will grow and replicate. Various cold-adapted human influenza A viruses, produced by reassortment with existing cold-adapted human influenza A viruses, have been shown to elicit good immune responses in vaccinated individuals, and certain live attenuated cold-adapted reassortant human influenza A viruses have proven to protect humans against challenge with wild-type virus. See, e.g., Clements, et al., 1986, J. Clin. Microbiol. 23, 73-76. In U.S. Pat. No. 5,149,531, by Youngner, et al., issued Sep. 22, 1992, the inventors of the present invention further demonstrated that certain reassortant cold-adapted human influenza A viruses also possess a dominant interference phenotype, i.e., they inhibit the growth of their corresponding parental wild-type strain, as well as heterologous influenza A viruses. U.S. Pat. No. 4,683,137, by Coggins et al., issued Jul. 28, 1987, and U.S. Pat. No. 4,693,893, by Campbell, issued Sep. 15, 1987, disclose attenuated therapeutic compositions produced by reassortment of wild type equine influenza viruses with attenuated, cold-adapted human influenza A viruses. Although these therapeutic compositions appear to be generally safe and effective in horses, they pose a significant danger of introducing into the environment a virus containing both human and equine influenza genes.

SUMMARY OF THE INVENTION

The present invention provides experimentally-generated cold-adapted equine influenza viruses, reassortant influenza A viruses that comprise at least one genome segment of an equine influenza virus generated by cold-adaptation such that the equine influenza virus genome segment confers at least one identifying phenotype of a cold-adapted equine influenza virus on the reassortant virus, and genetically-engineered equine influenza viruses, produced through reverse genetics, which comprise at least one identifying phenotype of a cold-adapted equine influenza virus. Identifying phenotypes include cold-adaptation, temperature sensitivity, dominant interference, and attenuation. The invention further provides a therapeutic composition to protect an animal against disease caused by an influenza A virus, where the therapeutic composition includes a cold-adapted equine influenza virus a reassortant influenza A virus, or a genetically-engineered equine influenza virus of the present invention. Also provided is a method to protect an animal from diseases caused by an influenza A virus which includes the administration of such a therapeutic composition. Also provided are methods to produce a cold-adapted equine influenza virus, and methods to produce a reassortant influenza A virus which comprises at least one genome segment of a cold-adapted equine influenza virus, where the equine influenza genome segment confers on the reassortant virus at least one identifying phenotype of the cold-adapted equine influenza virus.

A cold-adapted equine influenza virus is one that replicates in embryonated chicken eggs at a temperature ranging from about 26° C. to about 30° C. Preferably, a cold-adapted equine influenza virus, reassortant influenza A virus, or genetically-engineered equine influenza virus of the present invention is attenuated, such that it will not cause disease in a healthy animal.

In one embodiment, a cold-adapted equine influenza virus, reassortant influenza A virus, or genetically-engineered equine influenza virus of the present invention is also temperature sensitive, such that the virus replicates in embryonated chicken eggs at a temperature ranging from about 26° C. to about 30° C., forms plaques in tissue culture cells at a permissive temperature of about 34° C., but does not form plaques in tissue culture cells at a non-permissive temperature of about 39° C.

In one embodiment, such a temperature sensitive virus comprises two mutations: a first mutation that inhibits plaque formation at a temperature of about 39° C., that mutation co-segregating with the genome segment that encodes the viral nucleoprotein gene; and a second mutation that inhibits all viral protein synthesis at a temperature of about 39° C.

In another embodiment, a cold-adapted, temperature sensitive equine influenza virus of the present invention replicates in embryonated chicken eggs at a temperature ranging from about 26° C. to about 30° C., forms plaques in tissue culture cells at a permissive temperature of about 34° C., but does not form plaques in tissue culture cells or express late viral proteins at a non-permissive temperature of about 37° C.

Typically, a cold-adapted equine influenza virus of the present invention is produced by passaging a wild-type equine influenza virus one or more times, and then selecting viruses that stably grow and replicate at a reduced temperature. A cold-adapted equine, influenza virus produced thereby includes, in certain embodiments, a dominant interference phenotype, that is, the virus, when co-infected with a parental equine influenza virus or heterologous wild-type influenza A virus, will inhibit the growth of that virus.

Examples of cold-adapted equine influenza viruses of the present invention include EIV-P821, identified by accession No. ATCC VR-2625; EIV-P824, identified by accession No. ATCC: VR-2624; EIV-MSV+5, identified by accession No. ATCC VR-627; and progeny of such viruses.

Therapeutic compositions of the present invention include from about 10⁵ TCID₅₀ units to about 10⁸ TCID₅₀ units, and preferably about 2×10⁶ TCID₅₀ units, of a cold-adapted equine influenza virus, reassortant influenza A virus, or genetically-engineered equine influenza virus of the present invention.

The present invention also includes a method to protect an animal from disease caused by an influenza A virus, which includes the step of administering to the animal a therapeutic composition including a cold-adapted equine influenza virus, a reassortant influenza A virus, or a genetically-engineered equine influenza virus of the present invention. Preferred animals to protect include equids, with horses and ponies being particularly preferred.

Yet another embodiment of the present invention is a method to generate a cold-adapted equine influenza virus. The method includes the steps of passaging a wild-type equine influenza virus; and selecting viruses that grow at a reduced temperature. In one embodiment, the method includes repeating the passaging and selection steps one or more times, while progressively reducing the temperature. Passaging of equine influenza virus preferably takes place in embryonated chicken eggs.

Another embodiment is an method to produce a reassortant influenza A virus through genetic reassortment of the genome segments of a donor cold-adapted equine influenza virus of the present invention with the genome segments of a recipient influenza A virus. Reassortant influenza A viruses of the present invention are produced by a method that includes the steps of: (a) mixing the genome segments of a donor cold-adapted equine influenza virus with the genome segments of a recipient influenza A virus, and (b) selecting viruses which include at least one identifying phenotype of the donor equine influenza virus. Identifying phenotypes include cold-adaptation, temperature sensitivity, dominant interference, and attenuation. Preferably, such reassortant viruses at least include the attenuation phenotype of the donor virus. A typical reassortant virus will have the antigenicity of the recipient virus, that is, it will retain the hemagglutinin (HA) and neuraminidase (NA) phenotypes of the recipient virus.

The present invention further provides methods to propagate cold-adapted equine influenza viruses or reassortant influenza A viruses of the present invention. These methods include propagation in embryonated chicken eggs or in tissue culture cells.

The present invention also describes nucleic acid molecules encoding wild-type and cold-adapted equine influenza proteins M, HA, NS, PB2, PB2-N, PB2-C, PB1, PB1-N, PB1.-C, and PA-C. One embodiment of the present invention is an isolated equine nucleic acid molecule having a nucleic acid sequence selected from a group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25 SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:106 and SEQ ID NO:108 and a nucleic acid molecule comprising a nucleic acid sequence which is fully complementary to any of such nucleic acid sequences. Another embodiment of the present invention is an isolated equine nucleic acid molecule that encodes a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:77, SEQ ID NO:81, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:104 and SEQ ID NO:107. Another embodiment is an isolated equine influenza protein that comprises an amino acid sequence selected from a group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:77, SEQ ID NO:81, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:104 and SEQ ID NO:107. Also included in the present invention is a virus that include any of these nucleic acid molecules or proteins. In one embodiment, such a virus is equine influenza virus or a reassortant virus.

DETAILED DESCRIPTION

The present invention provides experimentally-generated cold-adapted equine influenza viruses comprising certain defined phenotypes, which are disclosed herein. It is to be noted that the term a or an entity, refers to one or more of that entity; for example, a cold-adapted equine influenza virus can include one or more cold-adapted equine influenza viruses. As such, the terms a (or an), one or more, and at least one can be used interchangeably herein. It is also to be noted that the terms comprising, including, and having can be used interchangeably. Furthermore, an item selected from the group consisting of refers to one or more of the items in that group, including combinations thereof.

A cold-adapted equine influenza virus of the present invention is a virus that has been generated in the laboratory, and as such, is not a virus as occurs in nature. Since the present invention also includes those viruses having the identifying phenotypes of such a cold-adapted equine influenza virus, an equine influenza virus isolated from a mixture of naturally-occurring viruses, i.e., removed from its natural milieu, but having the claimed phenotypes, is included in the present invention. A cold-adapted equine influenza virus of the present invention does not require any specific level of purity. For example, a cold-adapted equine influenza virus grown in embryonated chicken eggs may be in a mixture with the allantoic fluid (AF), and a cold-adapted equine influenza virus grown in tissue culture cells may be in a mixture with disrupted cells and tissue culture medium.

As used herein, an equine influenza virus is an influenza virus that infects and grows in equids, e.g., horses or ponies. As used herein, growth of a virus denotes the ability of the virus to reproduce or replicate itself in a permissive host cell. As such, the terms, growth of a virus and replication of a virus are used interchangeably herein. Growth or replication of a virus in a particular host cell can be demonstrated and measured by standard methods well-known to those skilled in the art of virology. For example, samples containing infectious virus, e.g., as contained in nasopharyngeal secretions from an infected horse, are tested for their ability to cause cytopathic effect (CPE), e.g., virus plaques, in tissue culture cells. Infectious virus may also be detected by inoculation of a sample into the allantoic cavity of embryonated chicken eggs, and then testing the AF of eggs thus inoculated for its ability to agglutinate red blood cells, i.e., cause hemagglutination, due to the presence of the influenza virus hemagglutinin (HA) protein in the AF.

Naturally-occurring, i.e., wild-type, equine influenza viruses replicate well at a temperature from about 34° C. to about 39° C. For example, wild-type equine influenza virus replicates in embryonated chicken eggs at a temperature of about 34° C., and replicates in tissue culture cells at a temperature from about 34° C. to about 39° C. As used herein, a cold-adapted equine influenza virus is an equine influenza virus that has been adapted to grow at a temperature lower than the optimal growth temperature for equine influenza virus. One example of a cold-adapted equine influenza virus of the present invention is a virus that replicates in embryonated chicken eggs at a temperature of about 30° C. A preferred cold-adapted equine influenza virus of the present invention replicates in embryonated chicken eggs at a temperature of about 28° C. Another preferred cold-adapted equine influenza virus of the present invention replicates in embryonated chicken eggs at a temperature of about 26° C. In general, preferred cold-adapted equine influenza viruses of the present invention replicate in embryonated chicken eggs at a temperature ranging from about 26° C. to about 30° C, i.e., at a range of temperatures at which a wild-type virus will grow poorly or not at all. It should be noted that the ability of such viruses to replicate within that temperature range does not preclude their ability to also replicate at higher or lower temperatures. For example, one embodiment is a cold-adapted equine influenza virus that replicates in embryonated chicken eggs at a temperature of about 26° C., but also replicates in tissue culture cells at a temperature of about 34° C. As with wild-type equine influenza viruses, cold-adapted equine influenza viruses of the present invention generally form plaques in tissue culture cells, for example Madin Darby Canine Kidney Cells (MDCK) at a temperature of about 34° C. Examples of suitable and preferred cold-adapted equine influenza viruses of the present invention are disclosed herein.

One embodiment of the present invention is a cold-adapted equine influenza virus that is produced by a method which includes passaging a wild-type equine influenza virus, and then selecting viruses that grow at a reduced temperature. Cold-adapted equine influenza viruses of the present invention can be produced, for example, by sequentially passaging a wild-type equine influenza virus in embryonated chicken eggs at progressively lower temperatures, thereby selecting for certain members of the virus mixture which stably replicate at the reduced temperature. An example of a passaging procedure is disclosed in detail in the Examples section. During the passaging procedure, one or more mutations appear in certain of the single-stranded RNA segments comprising the influenza virus genome, which alter the genotype, i.e., the primary nucleotide sequence of those RNA segments. As used herein, a mutation is an alteration of the primary nucleotide sequence of any given RNA segment making up an influenza virus genome. Examples of mutations include substitution of one or more nucleotides, deletion of one or more nucleotides, insertion of one or more nucleotides, or inversion of a stretch of two or more nucleotides. By selecting for those members of the virus mixture that stably replicate at a reduced temperature, a virus with a cold-adaptation phenotype is selected. As used herein, a phenotype is an observable or measurable characteristic of a biological entity such as a cell or a virus, where the observed characteristic is attributable to a specific genetic configuration of that biological entity, i.e., a certain genotype. As such, a cold-adaptation phenotype is the result of one or more mutations in the virus genome. As used herein, the terms a mutation, a genome, a genotype, or a phenotyperefer to one or more, or at least one mutation, genome, genotype, or phenotype, respectively.

Additional, observable phenotypes in a cold-adapted equine influenza virus may occur, and will generally be the result of one or more additional mutations in the genome of such a virus. For example, a cold-adapted equine influenza virus of the present invention may, in addition, be attenuated, exhibit dominant interference, and/or be temperature sensitive.

In one embodiment, a cold-adapted equine influenza virus of the present invention has a phenotype characterized by attenuation. A cold-adapted equine influenza virus is attenuated, when administration of the virus to an equine influenza virus-susceptible animal results in reduced or absent clinical signs in that animal, compared to clinical signs observed in animals that are infected with wild-type equine influenza virus. For example, an animal infected with wild-type equine influenza virus will display fever, sneezing, coughing, depression, and nasal discharges. In contrast, an animal administered an attenuated, cold-adapted equine influenza virus of the present invention will display minimal or no, i.e., undetectable, clinical disease signs.

In another embodiment, a cold-adapted equine influenza virus of the present invention comprises a temperature sensitive phenotype. As used herein, a temperature sensitive cold-adapted equine influenza virus replicates at reduced temperatures, but no longer replicates or forms plaques in tissue culture cells at certain higher growth temperatures at which the wild-type virus will replicate and form plaques. While not being bound by theory, it is believed that replication of equine influenza viruses with a temperature sensitive phenotype is largely restricted to the cool passages of the upper respiratory tract, and does not replicate efficiently in the lower respiratory tract, where the virus is more prone to cause disease symptoms. A temperature at which a temperature sensitive virus will grow is referred to herein as a permissive temperature for that temperature sensitive virus, and a higher temperature at which the temperature sensitive virus will not grow, but at which a corresponding wild-type virus will grow, is referred to herein as a non-permissive temperature for that temperature sensitive virus. For example, certain temperature sensitive cold-adapted equine influenza viruses of the present invention replicate in embryonated chicken eggs at a temperature at or below about 30° C., preferably at about 28° C. or about 26° C., and will form plaques in tissue culture cells at a permissive temperature of about 34° C., but will not form plaques in tissue culture cells at a non permissive temperature of about 39° C. Other temperature sensitive cold-adapted equine influenza viruses of the present invention replicate in embryonated chicken eggs at a temperature at or below about 30° C., preferably at about 28° C. or about 26° C., and will form plaques in tissue culture cells at a permissive temperature of about 34° C., but will not form plaques in tissue culture cells at a non-permissive temperature of about 37° C.

Certain cold-adapted equine influenza viruses of the present invention have a dominant interference phenotype; that is, they dominate an infection when co-infected into cells with another influenza A virus, thereby impairing the growth of that other virus. For example, when a cold-adapted equine influenza virus of the present invention, having a dominant interference phenotype, is co-infected into MDCK cells with the wild-type parental equine influenza virus, A/equine/Kentucky/1/91 (H3N8), growth of the parental virus is impaired. Thus, in an animal that has recently been exposed to, or may be soon exposed to, a virulent influenza virus, i.e., an influenza virus that causes disease symptoms, administration of a therapeutic composition comprising a cold-adapted equine influenza virus having a dominant interference phenotype into the upper respiratory tract of that animal will impair the growth of the virulent virus, thereby ameliorating or reducing disease in that animal, even in the absence of an immune response to the virulent virus.

Dominant interference of a cold-adapted equine influenza virus having a temperature sensitive phenotype can be measured by standard virological methods. For example, separate monolayers of MDCK cells can be infected with (a) a virulent wild-type influenza A virus, (b) a temperature sensitive, cold-adapted equine influenza virus, and (c) both viruses in a co-infection, with all infections done at multiplicities of infection (MOI) of about 2 plaque forming units (pfu) per cell. After infection, the virus yields from the various infected cells are measured by duplicate plaque assays performed at the permissive temperature for the cold-adapted equine influenza virus and at the non-permissive temperature of that virus. A cold adapted equine influenza virus having a temperature sensitive phenotype is unable to form plaques at its non-permissive temperature, while the wild-type virus is able to form plaques at both the permissive and non-permissive temperatures. Thus it is possible to measure the growth of the wild-type virus in the presence of the cold adapted virus by comparing the virus yield at the non-permissive temperature of the cells singly infected with wild-type virus to the yield at the non-permissive temperature of the wild-type virus in doubly infected cells.

Cold-adapted equine influenza viruses of the present invention are characterized primarily by one or more of the following identifying phenotypes: cold-adaptation, temperature sensitivity, dominant interference, and/or attenuation. As used herein, the phrase an equine influenza virus comprises the identifying phenotype(s) of cold-adaptation, temperature sensitivity, dominant interference, and/or attenuation refers to a virus having such a phenotype(s). Examples of such viruses include, but are not limited to, EIV-P8211, identified by accession No. ATCC VR-2625, EIV-P824, identified by accession No. ATCC VR-2624, and EIV-MSV+5, identified by accession No. ATCC VR-2627, as well as EIV-MSV0, EIV, MSV+1, EIV-MSV+2, EIV-MSV+3, and EIV-MSV+4. Production of such viruses is described in the examples. For example, cold-adapted equine influenza virus EIV-P821 is characterized by, i.e., has the identifying phenotypes of, (a) cold-adaptation, e.g., its ability to replicate in embryonated chicken eggs at a temperature of about 26° C.; (b) temperature sensitivity, e.g., its inability to form plaques in tissue culture cells and to express late gene products at a non-permissive temperature of about 37° C., and its inability to form plaques in tissue culture cells and to synthesize any viral proteins at a non-permissive temperature of about 39° C.; (c) its attenuation upon administration to an equine influenza virus-susceptible animal; and (d) dominant interference, e.g., its ability, when co-infected into a cell with a wild-type influenza A virus, to interfere with the growth of that wild-type virus. Similarly, cold-adapted equine influenza virus EIV-P824 is characterized by (a) cold adaptation, e.g., its ability to replicate in embryonated chicken eggs at a temperature of about 28° C.; (b) temperature sensitivity, e.g., its inability to form plaques in tissue culture cells at a non-permissive temperature of about 39° C.; and (c) dominant interference, e.g., its ability, when co-infected into a cell with a wild-type influenza A virus, to interfere with the growth of that wild-type virus. In another example, cold-adapted equine influenza virus EIV-MSV+5 is characterized by (a) cold-adaptation, e.g., its ability to replicate in embryonated chicken eggs at a temperature of about 26° C.; (b) temperature sensitivity, e.g., its inability to form plaques in tissue culture cells at a non permissive temperature of about 39° C.; and (c) its attenuation upon administration to an equine influenza virus-susceptible animal.

In certain cases, the RNA segment upon which one or more mutations associated with a certain phenotype occur may be determined through reassortment analysis by standard methods, as disclosed herein. In one embodiment, a cold-adapted equine influenza virus of the present invention comprises a temperature sensitive phenotype that correlates with at least two mutations in the genome of that virus. In this embodiment, one of the two mutations, localized by reassortment analysis as disclosed herein, inhibits, i.e., blocks or prevents, the ability of the virus to form plaques in tissue culture cells at a non-permissive temperature of about 39° C. This mutation co-segregates with the segment of the equine influenza virus genome that encodes the nucleoprotein (NP) gene of the virus, i.e., the mutation is located on the same RNA segment as the NP gene. In this embodiment, the second mutation inhibits all protein synthesis at a non-permissive temperature of about 39° C. As such, at the non-permissive temperature, the virus genome is incapable of expressing any viral proteins. Examples of cold-adapted equine influenza viruses possessing these characteristics are EIV-P821 and EIV MSV+5. EIV-P821 was generated by serial passaging of a wild-type equine influenza virus in embryonated chicken eggs by methods described in Example 1A. EIV-MSV+5 was derived by further serial passaging of EIV-P821, as described in Example 1E.

Furthermore, a cold-adapted, temperature sensitive equine influenza virus comprising the two mutations which inhibit plaque formation and viral protein synthesis at a non-permissive temperature of about 39° C. can comprise one or more additional mutations, which inhibit the virus” ability to synthesize late gene products and to form plaques in tissue culture cells at a non-permissive temperature of about 37° C. An example of la cold-adapted equine influenza virus possessing these characteristics is EIV-P821. This virus isolate replicates in embryonated chicken eggs at a temperature of about 26° C., and does not form plaques or express any viral proteins at a temperature of about 39° C. Furthermore, EIV-P821 does not form plaques on MDCK cells at a non-permissive temperature of about 37° C., and at this temperature, late gene expression is inhibited in such a way that late proteins are not produced, i.e., normal levels of NP protein are synthesized, reduced or undetectable levels of M1 or HA proteins are synthesized, and enhanced levels of the polymerase proteins are synthesized. Since this phenotype is typified by differential viral protein synthesis, it is distinct from the protein synthesis phenotype seen at a non-permissive temperature of about 39° C., which is typified by the inhibition of synthesis of all viral proteins.

Pursuant to 37 CFR §1.802 (a-c), cold-adapted equine influenza viruses, designated herein as EIV-P821, an EIV-P824 were deposited with the American Type Culture Collection (ATCC, 10801 University Boulevard, Manassas, Va. 20110-2209) under the Budapest Treaty as ATCC Accession Nos. ATCC VR-2625, and ATCC VR-2624, respectively, on Jul. 11, 1998. Cold-adapted equine influenza virus EIV-MSV+5 was deposited with the ATCC as ATCC Accession No. ATCC VR-2627on Aug. 3, 1998. Pursuant to 37 CFR §1.806, the deposits are made for a term of at least thirty (30) years and at least five (5) years after the most recent request for the furnishing of a sample of the deposit was received by the depository. Pursuant to 37 CFR §1.808 (a)(2), all restrictions imposed by the depositor on the availability to the public will be irrevocably removed upon the granting of the patent.

Preferred cold-adapted equine influenza viruses of the present invention have the identifying phenotypes of EIV-P821, EIV-P824, and EIV-MSV+5. Particularly preferred cold-adapted equine influenza viruses include EIV-P821, EIV-P824, EIV-MSV+5, and progeny of these viruses. As used herein, progeny are offspring, and as such can slightly altered phenotypes compared to the parent virus, but retain identifying phenotypes of the parent virus, for example, cold-adaptation, temperature sensitivity, dominant interference, or attenuation. For example, cold-adapted equine influenza virus EIV-MSV+5 is a progeny of cold-adapted equine influenza virus EIV-P821. Progeny also include reassortant influenza A viruses that comprise one or more identifying phenotypes of the donor parent virus.

Reassortant influenza A viruses of the present invention are produced by genetic reassortment of the genome segments of a donor cold-adapted equine influenza virus of the present invention with the genome segments of a recipient influenza A virus, and then selecting a reassortant virus that derives at least one of its eight RNA genome segments from the donor virus, such that the reassortant virus acquires at least one identifying phenotype of the donor cold-adapted equine influenza virus. Identifying phenotypes include cold-adaptation, temperature sensitivity, attenuation, and dominant interference. Preferably, reassortant influenza A viruses of the present invention derive at least the attenuation phenotype of the donor virus. Methods to isolate reassortant influenza viruses are well known to those skilled in the art of virology and are disclosed, for example, in Fields, et al., 1996, Fields Virology, 3d ed. Lippincott-Raven; and Palese, et al., 1976, J. ., 17, 876-884. Fields, et al., ibid. and Palese, et al., ibid.

A suitable donor equine influenza virus is a cold-adapted equine influenza virus of the present invention, for example, EIV-P821, identified by accession No. ATCC VR-2625, EIV-P824, identified by accession No. ATCC VR-2624, or EIV-MSV+5, identified by accession No. ATCTC VR-2627. A suitable recipient influenza A virus can be another equine influenza virus, for example a Eurasian subtype 2 equine influenza virus such as A/equine/Suffolk/89 (H3N8) or a subtype 1 equine influenza virus such as A/Prague/1/56 (H7N7). A recipient influenza A virus can also be any influenza A virus capable of forming a reassortant virus with a donor cold-adapted equine influenza virus. Examples of such influenza A viruses include, but are not limited to, human influenza viruses such as A/Puerto Rico/8/34 (H1N1), A/Hong Kong/156/97 (H5N1), A/Singapore/1/57 (H2N2), and A/Hong Kong/1/68 (H3N2); swine viruses such as A/Swine/Iowa/15/30 (1N1); and avian viruses such as A/mallard/New York/6750/78 (H2N2) and A/chicken/Hong Kong/258/97 (H5N1). A reassortant virus of the present invention can include any combination of donor and recipient gene segments, as long as the resulting reassortant virus possesses at least one identifying phenotype of the donor virus.

One example of a reassortant virus of the present invention is a 6+2″ reassortant virus, in which the six internal gene segments, i.e., those comprising the NP, PB2, PB1, PA, M, and NS genes, are derived from the donor cold-adapted equine influenza virus genome, and the two external gene segments, i.e., those comprising the HA and NA genes, are derived from the recipient influenza A virus. A resultant virus thus produced has the attenuated, cold-adapted, temperature sensitive, and/or dominant interference phenotypes of the donor cold-adapted equine influenza virus, but the antigenicity of the recipient strain.

In yet another embodiment, a cold-adapted equine influenza virus of the present invention can be produced through recombinant means. In this approach, one or more specific mutations, associated with identified cold-adaptation, attenuation, temperature sensitivity, or dominant interference phenotypes, are identified and are introduced back into a wild-type equine influenza virus strain using a reverse genetics approach. Reverse genetics entails using RNA polymerase complexes isolated from influenza virus-infected cells to transcribe artificial influenza virus genome segments containing the mutation(s), incorporating the synthesized RNA segment(s) into virus particles using a helper virus, and then selecting for viruses containing the desired changes. Reverse genetics methods for influenza viruses are described, for example, in Enami, et al., 1990, Proc. Natl. Acad. Sci. 87, 3802-3805; and in U.S. Pat. No. 5,578,473, by Palese, et al., issued Nov. 26, 1996. This approach allows one skilled in the art to produce additional cold-adapted equine influenza viruses of the present invention without the need to go through the lengthy cold-adaptation process, and the process of selecting mutants both in vitro and in vivo with the desired virus phenotype.

A cold-adapted equine influenza virus of the present invention may be propagated by standard virological methods well-known to those skilled in the art, examples of which are disclosed herein. For example, a cold-adapted equine influenza virus can be grown in embryonated chicken eggs or in eukaryotic tissue culture cells. Suitable continuous eukaryotic cell lines upon which to grow a cold-adapted equine influenza virus of the present invention include those that support growth of influenza viruses, for example, MDCK cells. Other suitable cells upon which to grow a cold-adapted equine influenza virus of the present invention include, but are not limited to, primary kidney cell cultures of monkey, calf, hamster or chicken.

In one embodiment, the present invention provides a therapeutic composition to protect an animal against disease caused by an influenza A virus, where the therapeutic composition includes either a cold-adapted equine influenza virus or a reassortant influenza A virus comprising at least one genome segment of an equine influenza virus generated by cold-adaptation, wherein the equine influenza virus genome segment confers at least one identifying phenotype of the cold-adapted equine influenza virus. In addition, a therapeutic composition of the present invention can include an equine influenza virus that has been genetically engineered to comprise one or more mutations, where those mutations have been identified to confer a certain identifying phenotype on a cold-adapted equine influenza virus of the present invention. As used herein, the phrase disease caused by an influenza A virus refers to the clinical manifestations observed in an animal which has been infected with a virulent influenza A virus. Examples of such clinical manifestations include, but are not limited to, fever, sneezing, coughing, nasal discharge, rales, anorexia and depression. In addition, the phrase disease caused by an influenza A virus is defined herein to include shedding of virulent virus by the infected animal. Verification that clinical manifestations observed in an animal correlate with infection by virulent equine influenza virus may be made by several methods, including the detection of a specific antibody and/or T-cell responses to equine influenza virus in the animal. Preferably, verification that clinical manifestations observed in an animal correlate with infection by a virulent influenza A virus is made by the isolation of the virus from the afflicted animal, for example, by swabbing the nasopharyngeal cavity of that animal for virus-containing secretions. Verification of virus isolation may be made by the detection of CPE in tissue culture cells inoculated with the isolated secretions, by inoculation of the isolated secretions into embryonated chicken eggs, where virus replication is dejectedly the ability of AF from the inoculated eggs to agglutinate erythrocytes, suggesting the presence of the influenza virus hemagglutinin protein, or by use of a commercially available diagnostic test, for example, the Directigen® FLU A test.

As used herein, the term to protect includes, for example, to prevent or to treat influenza A virus infection in the subject animal. As such, a therapeutic composition of the present invention can be used, for example, as a prophylactic vaccine to protect a subject animal from influenza disease by administering the therapeutic composition to that animal at some time prior to that animal's exposure to the virulent virus.

A therapeutic composition of the present invention, comprising a cold-adapted equine influenza virus having a dominant interference phenotype, can also be used to treat an animal that has been recently infected with virulent influenza A virus or is likely to be subsequently exposed in a few days, such that the therapeutic composition immediately interferes with the growth of the virulent virus, prior to the animal's production of antibodies to the virulent virus. A therapeutic composition comprising a cold-adapted equine influenza virus having a dominant interference phenotype may be effectively administered prior to subsequent exposure for a length of time corresponding to the approximate length of time that a cold-adapted equine influenza virus of the present invention will replicate in the upper respiratory tract of a treated animal, for example, up to about seven days. A therapeutic composition comprising a cold-adapted equine influenza virus having a dominant interference phenotype may be effectively administered following exposure to virulent equine influenza virus for a length of time corresponding to the time required for an infected animal to show disease symptoms, for example, up to about two days.

Therapeutic compositions of the present invention can be administered to any animal susceptible to influenza virus disease, for example, humans, swine, horses and other equids, aquatic birds, domestic and game fowl, seals, mink, and whales. Preferably, a therapeutic composition of the present invention is administered equids. Even more preferably, a therapeutic composition of the present invention is administered to a horse, to protect against equine influenza disease.

Current vaccines available to protect horses against equine influenza virus disease are not effective in protecting young foals, most likely because they cannot overcome the maternal antibody present in these young animals, and often, vaccination at an early age, for example 3 months of age, can lead to tolerance rather than immunity. In one embodiment, and in contrast to existing equine influenza virus vaccines, a therapeutic composition comprising a cold-adapted equine influenza virus of the present invention apparently can produce immunity in young animals. As such, a therapeutic composition of the present invention can be safely and effectively administered to young foals, as young as about 3 months of age, to protect against equine influenza disease without the induction of tolerance.

In one embodiment, a therapeutic composition of the present invention can be multivalent. For example, it can protect an animal from more than one strain of influenza A virus by providing a combination of one or more cold-adapted equine influenza viruses of the present invention, one or more reassortant influenza A viruses, and/or one or more genetically-engineered equine influenza viruses of the present invention. Multivalent therapeutic compositions can include at least two cold-adapted equine influenza viruses, e.g., against North American subtype-2 virus isolates such as A/equine/Kentucky/1/91 (H1N8), and Eurasian subtype-2 virus isolates such as A/equine/Suffolk/89 (H3N8); or one or more subtype-2 virus isolates and a subtype-1 virus isolate such as A/equine/Prague/1/56 (H7N7). Similarly, a multivalent therapeutic composition of the present invention can include a cold-adapted equine influenza virus and a reassortant influenza A virus of the present invention, or two reassortant influenza A viruses of the present invention. A multivalent therapeutic composition of the present invention can also contain one or more formulations to protect against one or more other infectious agents in addition to influenza A virus. Such other infectious agents include, but not limited to: viruses; bacteria; fungi and fungal-related microorganisms; and parasites. Preferable multivalent therapeutic compositions include, but are not limited to, a cold-adapted equine influenza virus, reassortant influenza A virus, or genetically-engineered equine influenza virus of the present invention plus one or more compositions protective against one or more other infectious agents that afflict horses. Suitable infectious agents to protect against include, but are not limited to, equine infectious anemia virus, equine herpes virus, eastern, western, or Venezuelan equine encephalitis virus, tetanus, Streptococcus equi, and Ehrlichia resticii.

A therapeutic composition of the present invention can be formulated in an excipient that the animal to be treated can tolerate. Examples of such excipients include water, saline, Ringer's solution, dextrose solution, Hank's solution, and other aqueous physiologically balanced salt solutions. Excipients can also contain minor amounts of additives, such as substances that enhance isotonicity and chemical or biological stability. Examples of buffers include phosphate buffer, bicarbonate buffer, and Tris buffer, while examples of stabilizers include A1/A2 stabilizer, available from Diamond Animal Health, Des Moines, Iowa. Standard formulations can either be liquids or solids which can be taken up in a suitable liquid as a suspension or solution for administration to an animal. In one embodiment, a non-liquid formulation may comprise the excipient salts, buffers, stabilizers, etc., to which sterile water or saline can be added prior to administration.

A therapeutic composition of the present invention may also include one or more adjuvants or carriers. Adjuvants are typically substances that enhance the immune response of an animal to a specific antigen, and carriers include those compounds that increase the half-life of a therapeutic composition in the treated animal. One advantage of a therapeutic composition comprising a cold-adapted equine influenza virus or a reassortant influenza A virus of the present invention is that adjuvants and carriers are not required to produce an efficacious vaccine. Furthermore, in many cases known to those skilled in the art, the advantages of a therapeutic composition of the present invention would be hindered by the use of some adjuvants or carriers. However, it should be noted that use of adjuvants or carriers is not precluded by the present invention.

Therapeutic compositions of the present invention include an amount of a cold-adapted equine influenza virus that is sufficient to protect an animal from challenge with virulent equine influenza virus. In one embodiment, a therapeutic composition of the present invention can include an amount of a cold-adapted equine influenza virus ranging from about 10⁵ tissue culture infectious dose-50 (TCID₅₀) units of virus to about 10⁸ TCID₅₀ units of virus. As used herein, a TCID₅₀ unit” is amount of a virus which results in cytopathic effect in 50% of those cell cultures infected. Methods to measure and calculate TCID₅₀ are known to those skilled in the art and are available, for example, in Reed and Muench, 1938, Am. J. of Hyg. 27, 493-497. A preferred therapeutic composition of the present invention comprises from about 10⁶ TCID₅₀ units to about 10⁷ TCID₅₀ units of a cold-adapted equine influenza virus or reassortant influenza A virus of the present invention. Even more preferred is a therapeutic composition comprising about 2×10⁶ TCID₅₀ units of a cold-adapted equine influenza virus or reassortant influenza A virus of the present invention.

The present invention also includes methods to protect an animal against disease caused by an influenza A virus comprising administering to the animal a therapeutic composition of the present invention. Preferred are those methods which protect an equid against disease caused by equine influenza virus, where those methods comprise administering to the equid a cold-adapted equine influenza virus. Acceptable protocols to administer therapeutic compositions in an effective manner include individual dose size, number of doses, frequency of dose administration, and mode of administration. Determination of such protocols can be accomplished by those skilled in the art, and examples are disclosed herein.

A preferable method to protect an animal against disease caused by an influenza A virus includes administering to that animal a single dose of a therapeutic composition comprising a cold-adapted equine influenza virus, a reassortant influenza A virus, or genetically-engineered equine influenza virus of the present invention. A suitable single dose is a dose that is capable of protecting an animal from disease when administered one or more times over a suitable time period. The method of the present invention may also include administering subsequent, or booster doses of a therapeutic composition. Booster administrations can be given from about 2 weeks to several years after the original administration. Booster administrations preferably are administered when the immune response of the animal becomes insufficient to protect the animal from disease. Examples of suitable and preferred dosage schedules are disclosed in the Examples section.

A therapeutic composition of the present invention can be administered to an animal by a variety of means, such that the virus will enter and replicate in the mucosal cells in the upper respiratory tract of the treated animal. Such means include, but are not limited to, intranasal administration, oral administration, and intraocular administration. Since influenza viruses naturally infect the mucosa of the upper respiratory tract, a preferred method to administer a therapeutic composition of the present invention is by intranasal administration. Such administration may be accomplished by use of a syringe fitted with cannula, or by use of a nebulizer fitted over the nose and mouth of the animal to be vaccinated.

The efficacy of a therapeutic composition of the present invention to protect an animal against disease caused by influenza A virus can be tested in a variety of ways including, but not limited to, detection of antibodies by, for example, hemagglutination inhibition (HAI) tests, detection of cellular immunity within the treated animal, or challenge of the treated animal with virulent equine influenza virus to determine whether the treated animal is resistant to the development of disease. In addition, efficacy of a therapeutic composition of the present invention comprising a cold-adapted equine influenza virus having a dominant interference phenotype to ameliorate or reduce disease symptoms in an animal previously inoculated or susceptible to inoculation with a virulent, wild-type equine influenza virus can be tested by screening for the reduction or absence of disease symptoms in the treated animal.

The present invention also includes methods to produce a therapeutic composition of the present invention. Suitable and preferred methods for making a therapeutic composition of the present invention are disclosed herein. Pertinent steps involved in producing one type of therapeutic composition of the present invention, i.e., a cold-adapted equine influenza virus, include (a) passaging a wild-type equine influenza virus in vitro, for example, in embryonated chicken eggs; (b) selecting viruses that grow at a reduced temperature; (c) repeating the passaging and selection steps one or more times, at progressively lower temperatures, until virus populations are selected which stably grow at the desired lower temperature; and (d) mixing the resulting virus preparation with suitable excipients.

The pertinent steps involved in producing another type of therapeutic composition of the present invent on, i.e., a reassortant influenza A virus having at least one genome segment of an equine influenza virus generated by adaptation, includes the steps of (a) mixing the genome segments of a donor cold-adapted equine influenza virus, which preferably also has the phenotypes of attenuation, temperature sensitivity, or dominant interference, with the genome segments of a recipient influenza A virus, and (b) selecting reassortant viruses that have at least one identifying phenotype of the donor equine influenza virus. Identifying phenotypes to select for include attenuation, cold-adaptation, temperature sensitivity, and dominant interference. Methods to screen for these phenotypes are well known to those skilled in the art, and are disclosed herein. It is preferable to screen for viruses that at least have the phenotype of attenuation.

Using this method to generate a reassortant influenza A virus having at least one genome segment of a equine influenza virus generated by cold-adaptation, one-type of reassortant virus to select for is a 6+2″ reassortant, where the six internal gene segments, i.e., those coding for the NP, PB2, PB1, PA, M, and NS genes, are derived from the donor cold adapted equine influenza virus genome, and the two external gene segments, i.e., those coding for the HA and NA genes, are derived from the recipient influenza A virus. A resultant virus thus produced can have the cold-adapted, attenuated, temperature sensitive, and/or interference phenotypes of the donor cold-adapted equine influenza virus, but the antigenicity of the recipient strain.

The present invention includes nucleic acid molecules isolated from equine influenza virus wild type strain A/equine/Kentucky/1/91 (H3N8), and cold-adapted equine influenza virus EIV-P821.

In accordance with the present invention, an isolated nucleic acid molecule is a nucleic acid molecule that has been removed from its natural milieu (i.e., that has been subject to human manipulation) and can include DNA, RNA, or derivatives of either DNA or RNA. As such, “isolated” does not reflect the extent to which the nucleic acid molecule has been purified.

The present invention includes nucleic acid molecules encoding wild-type and cold-adapted equine influenza virus proteins. Nucleic acid molecules of the present invention can be prepared by methods known to one skilled in the art. Proteins of the present invention can be prepared by methods known to one skilled in the art, i.e., recombinant DNA technology. Preferred nucleic acid molecules have coding strands comprising nucleic acid sequences SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:106 and SEQ ID NO:108 and/or a complement thereof. Complements are defined as two single strands of nucleic acid in which the nucleotide sequence is such that they will hybridize as a result of base pairing throughout their full length. Given a nucleotide sequence, one of ordinary skill in the art can deduce the complement.

Preferred nucleic acid molecules encoding equine influenza M proteins are nei_(wt)M₁₀₂₃, nei_(wt1)M₁₀₂₃, nei_(wt2)M₁₀₂₃, nei_(wt)M₇₅₆, nei_(wt1)M₇₅₆, nei_(wt2)M₇₅₆, nei_(ca1)M₁₀₂₃, nei_(ca2)M₁₀₂₃, nei_(ca1)M₇₅₆, and/or nei_(ca2)M₇₅₆, the coding strands of which are represented by SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, and/or SEQ ID NO:6.

Preferred nucleic acid molecules encoding equine influenza HA proteins are nei_(wt)HA₁₇₆₂, nei_(wt)HA₁₆₉₅, nei_(ca1)HA₁₇₆₂, nei_(ca2)HA₁₇₆₂, nei_(ca1)HA₁₆₉₅, and/or nei_(ca2)HA₁₆₉₅, the coding strands of which are represented by SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, and/or SEQ ID NO:12.

Preferred nucleic acid molecules encoding equine influenza PB2-N proteins are nei _(wt)PB2-N nei₁₂₄₁, nei_(wt)PB2-N₁₂₁₄, nei_(ca1)PB2-N₁₂₄₁nei_(ca2)PB2-N₁₂₄₁, nei_(ca1)PB2-N₁₂₁₄, nei_(ca2), and/or PB2-N₁₂₁₄, the coding strands of which are represented by SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, and/or SEQ ID NO:18.

Preferred nucleic acid molecules encoding equine influenza PB2-C proteins are nei_(wt1)PB2-C₁₂₃₃, nei_(wt2)PB2-C₁₂₂₃, nei_(wt)PB2-C₁₁₉₄, nei_(ca1)PB2-C₁₂₃₂, nei_(ca2)PB2-C₁₂₃₁, and/or nei_(ca1)PB2-C₁₁₉₄, the coding strands of which are represented by SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:21, SEQ ID NO:23, and/or SEQ ID NO:25.

Preferred nucleic, acid molecules encoding equine influenza PB2 proteins are nei_(wt)PB2₂₃₄₁, nei_(wt)PB2₂₂₇₇, nei_(ca1)PB2₂₃₄₁, and/or nei_(ca1)PB₂₂₇₇, the coding strands of which are represented by SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:47, and/or SEQ ID NO:49.

Preferred nucleic acid molecules encoding equine influenza NS proteins are nei_(wt1)NS₈₉₁, nei_(wt2)NS₈₉₁, nei_(wt1)NS₆₉₀, nei_(wt2)NS₆₉₀, nei_(wt3)NS₈₈₈, nei_(wt3)NS₆₉₀, nei_(wt4)NS₄₆₈, nei_(wt4)NS₂₉₃, nei_(ca1)NS₈₈₈, nei_(ca2)NS₈₈₈, nei_(ca1)NS₆₉₀, and/or nei_(ca2)NS₆₉₀ the coding strands of which are represented by SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:57 and/or SEQ ID NO:59.

Preferred nucleic acid molecules encoding equine influenza PB1-N proteins are nei_(wt1)PB1-N₁₂₂₉, nei_(wt1)PB1-N₁₁₉₄, nei_(wt2)PB1-N₆₇₃, nei_(wt2)PB1-N₆₃₆, nei_(ca1)PB1-N₁₂₂₅, nei_(ca1)PB1-N₁₁₈₅, nei_(ca2)PB1-N₁₂₂₁, and/or nei_(ca2)PB1-N₁₁₈₅ the coding strands of which are represented by SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:70, and/or SEQ ID NO:71.

Preferred nucleic acid molecules encoding equine influenza PA-C proteins are nei_(wt1)PA-C₁₂₂₈, nei_(wt1)PA-C₁₁₆₄, nei_(wt2)PA-C₁₂₂₃, nei_(wt2)PA-C₁₁₆₄, nei_(ca1)PA-C₁₂₃₃, nei_(ca2)PA-C₁₂₃₃, nei_(ca1)PA-C₁₁₇₀, and/or nei_(ca2)PA-C₁₁₇₀ the coding strands of which are represented by SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, and/or SEQ ID NO:82.

Preferred nucleic acid molecules encoding equine influenza PB1-C proteins are nei_(wt1)PB1-C₁₂₃₄, nei_(wt1)PB1-C₁₁₈₈, nei_(wt2)PB1-C₁₂₄₀, nei_(wt2)PB1-C₁₁₈₈, nei_(ca1)PB1-C₁₂₄₁, nei_(ca1)PB1-C₁₁₈₈, nei_(ca2)PB1-C₁₂₄₁ and/or nei_(ca2)PB1-C₁₁₈₈, the coding strands of which are represented by SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:94 and/or SEQ ID NO:96.

Preferred nucleic acid molecules encoding equine influenza PB1 proteins are nei_(wt)PB1₂₃₄₁, nei_(wt)PB1₂₂₇₁, nei_(ca1)PB1₂₃₄₁, nei_(ca1)PB1₂₂₇₁, the coding strands of which are represented by SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:106, and/or SEQ ID NO:108.

The present invention includes proteins comprising SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:77, SEQ ID NO:81, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:104 and SEQ ID NO:107 as well as nucleic acid molecules encoding such proteins.

Preferred equine influenza M proteins of the present invention include proteins encoded by a nucleic acid molecule comprising nei_(wt)M₁₀₂₃, nei_(wt1)M₁₀₂₃, nei_(wt2)M₁₀₂₃, nei_(wt)M₇₅₆, nei_(wt1)M₇₅₆, nei_(wt2)M₇₅₆, nei_(ca1)M₁₀₂₃, nei_(ca2)M₁₀₂₃, nei_(ca1)M₇₅₆, and/or nei_(ca2)M₇₅₆. Preferred equine influenza M proteins are Pei_(wt)M₂₅₂, Pei_(ca1)M₂₅₂, and/or Pei_(ca2)M₂₅₂. In one embodiment, a preferred equine influenza M protein of the present invention is encoded by SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, and/or SEQ ID NO:6, and, as such, has an amino acid sequence that includes SEQ ID NO:2 and/or SEQ ID NO:5.

Preferred equine influenza HA proteins of the present invention include proteins encoded by a nucleic acid molecule comprising nei_(wt)HA₁₇₆₂, nei_(wt)HA₁₆₉₅, nei_(ca1)HA₁₇₆₂, nei_(ca2)HA₁₇₆₂, nei_(ca1)HA₁₆₉₅, and/or nei_(ca2)HA₁₆₉₅. Preferred equine influenza HA proteins are P Pei_(wt)HA₅₆₅, Pei_(ca1)HA₅₆₅, and/or Pei_(ca2)HA₅₆₅. In one embodiment, a preferred equine influenza HA protein of the present invention is encoded by SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, and/or SEQ ID NO:12, and, as such, has an amino acid sequence that includes SEQ ID NO:8 and/or SEQ ID NO:11.

Preferred equine influenza PB2-N proteins of the present invention include proteins encoded by a nucleic acid molecule comprising nei_(wt)PB2-N₁₂₄₁, nei_(wt)PB2-N₁₂₁₄, nei_(ca1)PB2-N₁₂₄₁nei_(ca2)PB2-N₁₂₄₁, nei_(ca1)PB2-N₁₂₁₄nei_(ca2), and/or PB2-N₁₂₁₄. Preferred equine influenza PB2-N proteins are P_(wt)PB2-N₄₀₄, P_(ca1)PB2-N₄₀₄, and/or P_(ca2)PB2-N₄₀₄. In one embodiment, a preferred equine influenza PB2-N protein of the present invention is encoded by SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, and/or SEQ ID NO:18, and, as such, has an amino acid sequence that includes SEQ ID NO:14 and/or SEQ ID NO:17.

Preferred equine influenza PB2-C proteins of the present invention include proteins encoded by a nucleic acid molecule comprising nei_(wt1)PB2-C₁₂₃₃, nei_(wt2)PB2-C₁₂₃₂, nei_(wt)PB2-C₁₁₉₄, nei_(ca1)PB2-C₁₂₃₂, nei_(ca2)PB2-C₁₂₃₁, and/or nei_(ca1)PB2-C₁₁₉₄. Preferred equine influenza PB2-N proteins are P_(wt)PB2-C₃₉₈, P_(ca1)PB2-C₃₉₈, and/or P_(ca2)PB2-C₃₉₈. In one embodiment, a preferred equine influenza PB2-C protein of the present invention is encoded by SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:21, SEQ ID NO:23, and/or SEQ ID NO:25, and, as such, has an amino acid sequence that includes SEQ ID NO:20 and/or SEQ ID NO:24.

Preferred equine influenza PB2 proteins of the present invention include proteins encoded by a nucleic acid molecule comprising nei_(wt)PB2₂₃₄₁, nei_(wt)PB2₂₂₇₇, nei_(ca1)PB2₂₃₄₁, and or nei_(ca1)PB2₂₂₇₇. Preferred equine influenza PB2 proteins are Pei_(wt)PB2₇₅₉, and/or Pei_(ca1)PB2₇₅₉. In one embodiment, a preferred equine influenza PB2 protein of the present invention is encoded by SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:47, and/or SEQ ID NO:49, and, as such, has an amino acid sequence that includes SEQ ID NO:45 and/or SEQ ID NO:48.

Preferred equine;influenza NS proteins of the present invention include proteins encoded by a nucleic acid molecule comprising nei_(wt1)NS₈₉₁, nei_(wt2)NS₈₉₁, nei_(wt1)NS₆₉₀, nei_(wt3)NS₈₈₈, nei_(wt4)NS₄₆₈, nei_(wt4)NS₂₉₃, nei_(ca1)NS₈₈₈, nei_(ca2)NS₈₈₈, and/or nei_(ca1)NS₆₉₀. Preferred equine influenza NS proteins are Pei_(wt)NS₂₃₀, Pei_(wt4)NS₉₇, and/or Pei_(ca1)NS₂₃₀. In one embodiment, a preferred equine influenza NS protein of the present invention is encoded by SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:57 and/or SEQ ID NO:59, and, as such, has an amino acid sequence that includes SEQ ID NO:51, SEQ ID NO:55 and/or SEQ ID NO:58.

Preferred equine influenza PB1-N proteins of the present invention include proteins encoded by a nucleic acid molecule comprising nei_(wt1)PB1-N₁₂₂₉, nei_(wt1)PB1N₁₁₉₄, nei_(wt2)PB1-N₆₇₃, nei_(wt2)PB1-N₆₃₆, nei_(ca1)PB21-N₁₂₂₅, nei_(ca1)PB1-N₁₁₈₅, and/or nei_(ca2)PB1-N₁₂₂₁. Preferred equine influenza PB1-N proteins are Pei_(wt1)PB1-N₃₉₈, P_(wt2)PB1-N₂₁₂, and/or P_(ca1)PB1-N₃₉₅. In one embodiment, a prefer red equine influenza PB1-N protein of the present invention is encoded by SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:70, and/or SEQ ID NO:71, and, as such, has an amino acid sequence that includes SEQ ID NO:63, SEQ ID NO:66 and/or SEQ ID NO:69.

Preferred equine influenza PB1-C proteins of the present invention include proteins encoded by a nucleic acid molecule comprising nei_(wt1)PB1-C₁₂₃₄, nei_(wt1)PB1-C₁₁₈₈, nei_(wt2)PB1-C₁₂₄₀, nei_(wt2)PB1-C₁₁₈₈, nei_(ca1)PB1-C₁₂₄₁, nei_(ca1)PB1-C₁₁₈₈, nei_(ca2)PB1-C₁₂₄₁ and/or nei_(ca2)PB1-C₁₁₈₈. Preferred equine influenza PB1-C proteins are Pei_(wt1)PB1-C₃₉₆, Pei_(wt2)PB1-C₃₉₆Pei_(ca1)PB1-C₃₉₆, and/or Pei_(ca2)PB1-C₃₉₆. In one embodiment, a preferred equine influenza PB1-C protein of the present invention is encoded by SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:94, and/or SEQ ID NO:96, and, as such, has an amino acid sequence that includes SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO 92, and/or SEQ ID NO:95.

Preferred equine influenza PB1 proteins of the present invention include proteins encoded by a nucleic, acid molecule comprising nei_(wt)PB₂₃₄₁, nei_(wt)PB1₂₂₇₁, nei_(ca1)PB1₂₃₄₁, nei_(ca1)PB1₂₂₇₁. Preferred equine influenza PB1 proteins are Pei_(wt)PB1₇₅₇, and/or Pei_(ca1)PB1₇₅₇. In one embodiment, a preferred equine influenza PB1 protein of the present invention is encoded by SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:106, and/or SEQ ID NO:108, and, as such, has an amino acid sequence that includes SEQ ID NO:104 and/or SEQ ID NO:107.

Preferred equine, influenza PA-C proteins of the present invention include proteins encoded by a nucleic acid molecule comprising nei_(wt1)PA-C₁₂₂₈, nei_(wt1)PA-C₁₁₆₄, nei_(wt2)PA-C₁₂₂₃, nei_(ca1)PA-C₁₂₃₃, nei_(ca2)PA-C₁₂₃₃, and/or nei_(ca1)PA-C₁₁₇₀. Preferred equine influenza PA-C proteins are Pei_(wt1)PA-C₃₈₈, and/or Pei_(ca1)PA-C₃₉₀. In one embodiment, a preferred equine influenza PA-C protein of the present invention is encoded by SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, and/or SEQ ID NO:82, and, as such, has an amino acid sequence that includes SEQ ID NO:77 and/or SEQ ID NO:81.

Nucleic acid sequence SEQ ID NO:1 represents the consensus sequence deduced from the coding strand of PCR amplified nucleic acid molecules denoted herein as nei_(wt1)M₁₀₂₃ and nei_(wt2)M₁₀₂₃, the production of which is disclosed in the Examples. Nucleic acid sequence SEQ ID NO:4 represents the deduced sequence of the coding strand of PCR amplified nucleic acid molecules denoted herein as nei_(ca1)M₁₀₂₃ and nei_(ca2)M₁₀₂₃, the production of which is disclosed in the Examples. Nucleic acid sequence SEQ ID NO:7 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as nei_(wt)HA₁₇₆₂, the production of which is disclosed in the Examples. Nucleic acid sequence SEQ ID NO:10 represents the deduced sequence of the coding strand of PCR amplified nucleic acid molecules denoted herein as nei_(ca1)HA₁₇₆₂ and nei_(ca2)HA₁₇₆₂, the production of which is disclosed in the Examples. Nucleic acid sequence SEQ ID NO:13 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as nei_(wt)PB2-N₁₂₄₁, the production of which is disclosed in the Examples. Nucleic acid sequence SEQ ID NO:16 represents the deduced sequence of the coding strand of PCR amplified nucleic acid molecules denoted herein as nei_(ca1)PB2-N₁₂₄₁ and nei_(ca2)PB2-N₁₂₄₁, the production of which is disclosed in the Examples. Nucleic acid sequence SEQ ID NO:19 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as nei_(wt1)PB2-C₁₂₃₃, the production of which is disclosed in the examples. Nucleic acid sequence SEQ ID NO:22 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as nei_(wt2)PB2-C₁₂₃₂, the production of which is disclosed in the examples. Nucleic acid sequence SEQ ID NO:23 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as nei_(ca1)PB2-C₁₂₃₂, the production of which is disclosed in the examples. Nucleic acid sequence SEQ ID NO:44 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as nei_(wt)PB2₂₃₄₁ the production of which is disclosed in the Examples. Nucleic acid sequence SEQ ID NO:47 represents the deduced sequence of the coding strand of PCR amplified nucleic acid molecules denoted herein as nei_(ca1)PB2₂₃₄₁ the production of which is disclosed in the Examples. Nucleic acid sequence SEQ ID NO:50 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as nei_(wt1)NS₈₉₁ and nei_(wt2)NS₈₉₁the production of which is disclosed in the examples. Nucleic acid sequence SEQ ID NO:53 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as nei_(wt3)NS₈₈₈, the production of which is disclosed in the examples. Nucleic acid sequence SEQ ID NO:54 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as nei_(wt4)NS₄₆₈, the production of which is disclosed in the examples. Nucleic acid sequence SEQ ID NO:57 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as nei_(ca1)NS₈₈₈ and nei_(ca1)NS₈₈₇, the production of which is disclosed in the Examples. Nucleic acid sequence SEQ ID NO:62 represents the deduced sequence of the coding strand of PCR amplified nucleic acid molecules denoted herein as nei_(wt1)PB1-N₁₂₂₉, the production of which is disclosed in the Examples. Nucleic acid sequence SEQ ID NO:65 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as nei_(wt2)PB2-N₆₇₃, the production of which is disclosed in the examples. Nucleic acid sequence SEQ ID NO:68 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as nei_(ca1)PB1-N₁₂₂₅, the production of which is disclosed in the examples. Nucleic acid sequence SEQ ID NO:71 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as nei_(ca2)PB1-N₁₂₂₁, the production of which is disclosed in the examples. Nucleic acid sequence SEQ ID NO:76 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as nei_(wt1)PA-C₁₂₂₈, the production of which is disclosed in the examples. Nucleic acid sequence SEQ ID NO:79 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as nei_(wt2)PA-C₁₂₂₃, the production of which is disclosed in the examples. Nucleic acid sequence SEQ ID NO:80 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as nei_(ca1)PA-C₁₂₃₃ and nei_(ca2)PA-C₁₂₃₃ the production of which is disclosed in the examples. Nucleic acid sequence SEQ ID NO:85 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as nei_(ca1)PB1-C₁₂₃₄ the production of which is disclosed in the examples. Nucleic acid sequence SEQ ID NO:88 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as nei_(wt2)PB1-C₁₂₄₀ the production of which is disclosed in the examples. Nucleic acid sequence SEQ ID NO:91 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as nei_(ca1)PB1-C₁₂₄₁ the production of which is disclosed in the examples. Nucleic acid sequence SEQ ID NO:94 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as nei_(ca2)PB1-C₁₂₄₁ the production of which is disclosed in the examples. Nucleic acid sequence SEQ ID NO:103 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as nei_(wt)PB1₂₃₄₁ the production of which is disclosed in the examples. Nucleic acid sequence SEQ ID NO:105 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as nei_(wt)PB1₂₂₇₁ the production of which is disclosed in the examples. Nucleic acid sequence SEQ ID NO:106 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as nei_(ca)PB1₂₃₄₁ the production of which is disclosed in the examples. Nucleic acid sequence SEQ ID NO:108 represents the deduced sequence of the coding strand of a PCR amplified nucleic acid molecule denoted herein as nei_(ca)PB1₂₂₇₁ the production of which is disclosed in the examples. Additional nucleic acid molecules, nucleic acid sequences, proteins and amino acid sequences are described in the Examples.

The present invention includes nucleic acid molecule comprising a cold-adapted equine influenza virus encoding an M protein having an amino acid sequence comprising SEQ ID NO:5. Another embodiment of the present invention includes a nucleic acid molecule comprising a cold-adapted equine influenza virus encoding an HA protein having an amino acid sequence comprising SEQ ID NO:11. Another embodiment of the present invention includes a nucleic acid molecule comprising a cold-adapted equine influenza virus encoding a PB2-N protein having an amino acid sequence comprising SEQ ID NO:17. Another embodiment of the present invention includes a nucleic acid molecule comprising a cold-adapted equine influenza virus encoding a PB2-C protein having an amino acid sequence comprising SEQ ID NO:24. Another embodiment of the present invention includes a nucleic acid molecule comprising a cold-adapted equine influenza virus encoding a PB protein having an amino acid sequence comprising SEQ ID NO:48. Another embodiment of the present invention includes a nucleic acid molecule comprising a cold-adapted equine influenza virus encoding a NS protein having an amino acid sequence comprising SEQ ID NO:58. Another embodiment of the present invention includes a nucleic acid molecule comprising a cold-adapted equine influenza virus encoding a PB1-N protein having an amino acid sequence comprising SEQ ID NO:69. Another embodiment of the present invention includes a nucleic acid molecule comprising a cold-adapted equine influenza virus encoding a PA-C protein having an amino acid sequence comprising SEQ ID NO:81. Another embodiment of the present invention includes a nucleic acid molecule comprising a cold-adapted equine influenza virus encoding a PB1-C protein having an amino acid sequence comprising SEQ ID NO:92. Another embodiment of the present invention includes a nucleic acid molecule comprising a cold-adapted equine influenza virus encoding a PB1 protein having an amino acid sequence comprising SEQ ID NO:107.

It should be noted that since nucleic acid sequencing technology is not entirely error-free, the nucleic acid sequences and amino acid sequences presented herein represent, respectively, apparent nucleic acid sequences of nucleic acid molecules of the present invention and apparent amino acid sequences of M, HA, PB2-N, PB2-C, PB2, NS, PB1-N, PA-C, PB1-C and PB1 proteins of the present invention.

Another embodiment of the present invention is an antibody that selectively binds to an wild-type virus M, HA, PB2-N, PB2-C, PB2, NS, PB1-N, PA-C, PB1-C and PB1 protein of the present invention. Another embodiment of the present invention is an antibody that selectively binds to a cold-adapted virus M, HA, PB2-N, PB2-C, PB2, NS, PB1-N, PA-C, PB1-C and PB1 protein of the present invention. Preferred antibodies selectively bind to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:69, SEQ ID NO:77, SEQ ID NO:81, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:104 and SEQ ID NO:107.

The following examples are provided for the purposes of illustration and are not intended to limit the scope of the present invention.

EXAMPLE 1

This example discloses the production and phenotypic characterization of several cold-adapted equine influenza viruses of the present invention.

A. Parental equine influenza virus, A/equine/Kentucky/1/91 (H3N8) (obtained from Tom Chambers, the University of Kentucky, Lexington, Ky.) was subjected to cold-adaptation in a,foreign host species, i.e., embryonated chicken eggs, in the following manner. Embryonated, 10 or 11-day old chicken eggs, available, for example, from Truslow Farms, Chestertown, Md. or from HyVac, Adel, Iowa, were inoculated with the parental equine influenza virus by injecting about 0.1 milliliter (ml) undiluted AF containing approximately 10⁶ plaque forming units (pfu) of virus into the allantoic cavity through a small hole punched in the shell of the egg. The holes in the eggs were sealed with nail polish and the eggs were incubated in a humidified incubator set at the appropriate temperature for three days. Following incubation, the eggs were candied and any non-viable eggs were discarded. AF was harvested from viable embryos by aseptically removing a portion of the egg shell, pulling aside the chorioallantoic membrane (CAM) with sterile forceps and removing the AF with a sterile pipette. The harvested AF was frozen between passages. The AF was then used, either undiluted or diluted 1:1000 in phosphate-buffered saline (PBS) as noted in Table 1, to inoculateda new set of eggs for a second passage, and so on. A total of 69 passages were competed. Earlier passages were done at either about 34° C. (passages 1-2) or about 30° C. and on subsequent passages, the incubation temperature was shifted down either to about 28° C., or to about 26° C. In order to increase the possibility of the selection of the desired phenotype of a stable, attenuated virus, the initial serial passage was expanded to included five different limbs of the serial passage tree, A through E, as shown in Table 1.

TABLE 1 Passage history of the limbs A through E. [t1] Passage # Temperature Limb A Limb B Limb C Limb D Limb E 341 C. 1-2  1-2  1-2  1-2  1-2 301 C. 3-8  3-29  3-29  3-29  3-29 281 C. 30-33* 30-68* 30-33 30-69 261 C. 9-65 34-69* 34-65 *= the infectious allantoic fluid was diluted 1:1000 in these passages.

B. Virus isolates carried through the cold-adaptation procedure described in section A were tested: for temperature sensitivity, i.e., a phenotype in which the cold-adapted virus grows at the lower, or permissive temperature (e.g., about 34° C.), but no longer forms plaques at a higher, or non-permissive temperature (e.g., about 37° C. or about 39° C.), as follows. At each cold-adaptation passage, the AF was titered by plaque assay at about 34° C. Periodically, individual plaques from the assay were clonally isolated by excision of the plaque area and placement of the excised agar plug in a 96-well tray containing a monolayer of MDCK cells. The 96-well trays were incubated overnight and the yield assayed for temperature sensitivity by CPE assay in duplicate 96-well trays incubated at about 34° C. and at about 39° C. The percent of the clones that scored as temperature sensitive mutants by this assay, i.e., the number of viral plaques that grew at 34° C. but did not grow at 39° C., divided by the total number of plaques, was calculated, and is shown in Table 2. Temperature sensitive isolates were then evaluated for protein synthesis at the non-permissive temperature by visualization of radiolabeled virus-synthesized proteins by SDS polyacrylamide gel electrophoresis (SDS-PAGE).

TABLE 2 Percent of isolated Clones that were temperature sensitive. [t21] Percent Temperature Sensitive Passage # Limb A Limb B Limb C Limb D Limb E p36  56% 66%  0% 66% 54% p46  80%  60% 75% p47  80% p48 100% p49 100% 100% 50% p50  90% p51 100% p52 57% p62 100% 100% p65 100% p66 100% 88%

From the clonal isolates tested for temperature sensitivity, two were selected for further study. Clone EIV-P821 was selected from the 49th passage of limb B and clone EIV-P824 was selected, from the 48th passage of limb C, as defined in Table 1. Both of these virus isolates were temperature sensitive, with plaque formation of both isolates inhibited at a temperature of about 39° C. At this temperature, protein synthesis was completely inhibited by EIV-P821, but EIV-P824 exhibited normal levels of protein synthesis. In addition, plaque formation by EIV-P821 was inhibited at a temperature of about 37° C., and at this temperature, late gene expression was inhibited, i.e., normal levels of NP protein were synthesized, reduced or no M1 or HA proteins were synthesized, and enhanced levels of the polymerase proteins were synthesized. The phenotype observed at 37° C., being typified by differential viral protein synthesis, was distinct from the protein synthesis phenotype seen at about 39° C., which was typified by the inhibition of synthesis of all viral proteins. Virus EIV-P821 has been deposited with the American Type Culture Collection (ATCC) under Accession No. ATCC VR-2625, and virus EIV-P824 has been deposited with the ATCC under Accession No. ATCC VR-2624.

C. Further characterization of the mutations in isolate EIV-P821 were carried out by reassortment analysis, as follows. Reassortment analysis in influenza viruses allows one skilled in the art, under certain circumstances, to correlate phenotypes of a given virus with putative mutations occurring on certain of the eight RNA segments that comprise an influenza A virus genome. This technique is described, for example, in Palese, et al., ibid. A mixed infection of EIV-P821 and an avian influenza virus, A/mallard/New York/6750/78 was performed as follows. MDCK cells were co-infected with EIV-P821 at a multiplicity of infection (MOI) of 2 pfu/cell and A/mallard/New York/6750/78 at an MOI of either 2, 5, or 10 pfu/cell. The infected cells were incubated at a temperature of about 34° C. The yields of the various co-infections were titered and individual plaques were isolated at about 34° C., and the resultant clonal isolates were characterized as to whether they were able to grow at about 39° C. and about 37° C., and express their genes, i.e., synthesize viral proteins, at about 39° C., about 37° C., and about 34° C. Protein synthesis was evaluated by SDS-PAGE analysis of radiolabeled infected-cell lysates. The HA, NP and NS-1 proteins of the two parent viruses, each of which is encoded by a separate genome segment, were distinguishable by SDS-PAGE analysis, since these particular viral proteins, as derived from either the equine or the avian influenza virus, migrate at different apparent molecular weights. In this way it was possible, at least for the HA, NP, and NS-1 genes, to evaluate whether certain phenotypes of the parent virus, e.g., the temperature sensitive and the protein synthesis phenotypes, co-segregate with the genome segments carrying these genes. The results of the reassortment analyses investigating co-segregation of a) the mutation inhibiting plaque formation, i.e., the induction of CPE, at a non-permissive temperature of about 39° C. or b) the mutation inhibiting protein synthesis at a non-permissive temperature of about 39° C. with each of the EIV-P821 HA, NP and NS-1 proteins are shown in Tables 3 and 4, respectively.

TABLE 3 Reassortment analysis of the EIV-P821 39° C. plaque formation phenotype with avian influenza virus, A/mallard/New York/6750/78. [t17] Gene Virus ts+¹ ts−² HA avian 26 13 equine 11 44 NP avian 37 8 equine  0 49 NS-1 avian 9 8 equine 12 20 ¹number of clonal isolates able to induce CPE in tissue culture cells at a temperature of about 39° C. ²number of clonal isolates inhibited in the ability to induce CPE in tissue culture cells at a temperature of about 39° C.

TABLE 4 Reassortment analysis of the EIV-P821 39° C. protein synthesis phenotype with avian influenza virus, A/mallard/New York/6750/78. [t3] Gene Virus ts+¹ ts−² HA avian 18 1 equine 11 7 NP avian 34 5 equine  7 8 NS-1 avian 10 4 equine 14 5 ¹number of clonal isolates which synthesize all viral proteins at a temperature of about 39° C. ²number of clonal isolates inhibited in the ability to synthesize all viral proteins at a temperature of about 39° C.

The results demonstrated an association of the equine NP gene with a mutation causing the inability of EIV-P821 to form plaques at a non-permissive temperature of about 39° C., but the results did not suggest an association of any of the HA, NP, or NS-1 genes with a mutation causing the inability of EIV-P821 to express viral proteins at a non-permissive temperature of about 39° C. Thus, these data also demonstrated that the plaque formation phenotype and the protein synthesis phenotype observed in virus EIV-P821 were the result of separate mutations.

D. Studies were also conducted to determine if cold-adapted equine influenza viruses of the present invention have a dominant interference phenotype, that is, whether they dominate in mixed infection with the wild type parental virus A/Kentucky/1/91 (H:3N8). The dominant interference phenotype of viruses EIV-P821 and EIV-P824 were evaluated in the following manner. Separate monolayers of MDCK cells were singly infected with the parental virus A/Kentucky/1/91 (H3N8) at an MOI of 2, singly infected with either cold-adapted virus EIV-P821 or EIV-P824 at an MOI of 2, or simultaneously doubly infected with both the parental virus and one of the cold adapted viruses at an MOI of 2+2, all at a temperature of about 34° C. At 24 hours after infection, the media from the cultures were harvested and the virus yields from the various infected cells were measured by duplicate plaque assays performed at temperatures of about 34° C. and about 39° C. This assay took advantage of the fact that cold adapted equine influenza viruses EIV-P821 or EIV-P824 are temperature sensitive and are thus unable to form plaques at a non-permissive temperature of about 39° C., while the parental virus is able to form plaques at both temperatures, thus making it possible to measure the growth of the parental virus in the presence of the cold adapted virus. Specifically, the dominant interference effect of the cold adapted virus on the growth of the parental virus was quantitated by comparing the virus yield at about 39° C. of the cells singly infected with parental virus to the yield of the parental virus in doubly infected cells. EIV-P821, in mixed infection, was able to reduce the yield of the parental virus by approximately 200 fold, while EIV-P824, in mixed infection, reduced the yield of the parental virus by approximately 3200 fold. This assay therefore showed that cold-adapted equine influenza viruses EIV-P821 and EIV-P824 both exhibit the dominant interference phenotype.

E. Virus isolate EIV-MSV+5 was derived from EIV-P821, as follows. EIV-P821 was passaged once in eggs, as described above, to produce a Master Seed Virus isolate, denoted herein as EIV-MSV0. EIV-MSV0 was then subjected to passage three additional times in eggs, the virus isolates at the end of each passage being designated EIV-MSV+1, EIV-MSV+2, and EIV-MSV+3, respectively. EIV-MSV+3 was then subjected to two additional passages in MDCK cells, as follows. MDCK cells were grown in 150 cm² tissue culture flasks in MEM tissue culture medium with Hanks Salts, containing 10% calf serum. The cells were then washed with sterile PBS and the growth medium was replaced with about 8 ml per flask of infection medium (tissue culture medium comprising MEM with Hanks Salts, 1 μg/ml TPCK trypsin solution, 0.125% bovine serum albumin (BSA), and 10 mM HEPES buffer). MDCK cells were inoculated with AF containing virus EIV-MSV+3 (for the first passage in MDCK cells) or virus stock harvested from EIV-MSV+4 (for the second passage in MDCK cells), and the viruses were allowed to adsorb for 1 hour at about 34° C. The inoculum was removed from the cell monolayers, the cells were washed again with PBS, and about 100 ml of infection medium was added per flask. The infected cells were incubated at about 34° C. for 24 hours. The virus-infected MDCK cells were harvested by shaking the flasks vigorously to disrupt the cell monolayer, resulting in virus isolates EIV-MSV+4 (the first passage in MDCK cells), and EIV-MSV+5 (the second passage in MDCK cells). Viruses EIV-MSV0 and EIV-MSV+5 were subjected to phenotypic analysis, as described in section B above, to determine their ability to form plaques and synthesize viral proteins at temperatures of about 34° C., about 37° C., and about 39° C. Both EIV-MSV0 and EIV-MSV+5 formed plaques in tissue culture cells at a temperature of about 34° C., and neither virus isolate formed plaques or exhibited detectable viral protein synthesis at a temperature of about 39° C. Virus EIV-MSV0 had a similar temperature sensitive phenotype as EIV-P821 at a temperature of about 37° C., i.e., it was inhibited in plaque formation, and late gene expression was inhibited. However, EIV-MSV+5, unlike its parent virus, EIV-P821, did form plaques in tissue culture at a temperature of about 37° C., and at this temperature, the virus synthesized normal amounts of all proteins. Virus EIV-MSV+5 has been deposited with the ATCC under Accession No. ATCC VR-2627.

EXAMPLE 2

Therapeutic compositions of the present invention were produced as follows.

A. A large stock of EIV-P821 was propagated in eggs as follows. About 60 specific pathogen-free embryonated chicken eggs were candled and non-viable eggs were discarded. Stock virus was diluted to about 1.0×10⁵ pfu/ml in sterile PBS. Virus was inoculated into the allantoic cavity of the eggs as described in Example 1A. After a 3-day incubation in a humidified chamber at a temperature of about 34° C., AF was harvested from the eggs according to the method described in Example 1A. The harvested AF was mixed with a stabilizer solution, for example A1/A2 stabilizer, available from Diamond Animal Health, Des Moines, Iowa, at 25% V/V (stabilizer/AF). The harvested AF was batched in a centrifuge tube and was clarified by centrifugation for 10 minutes at 1000 rpm in an IEC Centra-7R refrigerated table top centrifuge fitted with a swinging bucket rotor. The clarified fluid was distributed into 1-ml cryovials and was frozen at about −70° C. Virus stocks were titrated on MDCK cells by CPE and plaque assay at about 34° C.

B. A large stock of EIV-P821 was propagated in MDCK cells as follows. MDCK cells were grown in 150 cm² tissue culture flasks in MEM tissue culture medium with Hanks Salts, containing 10% calf serum. The cells were then washed with sterile PBS and the growth medium was replaced with about 8 ml per flask of infection medium. The MDCK cells were inoculated with virus stock at an MOI ranging from about 0.5 pfu per cell to about 0.005 pfu per cell, and the viruses were allowed to adsorb for 1 hour at about 34° C. The inoculum was removed from the cell monolayers, the cells were washed again with PBS, and about 100 ml of infection medium was added per flask. The infected cells were incubated at about 34° C. for 24 hours. The virus-infected MDCK cells were harvested by shaking the flasks vigorously to disrupt the cell monolayer and stabilizer solution was added to the flasks at 25% V/V (stabilizer/virus solution). The supernatants were distributed aseptically into cryovials and frozen at −70° C.

C. Therapeutic compositions comprising certain cold-adapted temperature sensitive equine influenza viruses of the present invention were formulated as follows. Just prior to vaccination procedures, such as those described in Examples 3-7 below, stock vials of EIV-P821 or EIV-MSV+5 were thawed and were diluted in an excipient comprising either water, PBS, or in MEM tissue culture medium with Hanks Salts, containing 0.125% bovine serum albumin (BSA-MEM solution) to the desired dilution for administration to animals. The vaccine compositions were held on ice prior to vaccinations. All therapeutic compositions were titered on MDCK cells by standard methods just prior to vaccinations and wherever possible, an amount of the composition, treated identically to those administered to the animals, was titered after the vaccinations to ensure that the virus remained viable during the procedures.

EXAMPLE 3

A therapeutic composition comprising cold-adapted equine influenza virus EIV-P821 was tested for safety and its ability to replicate in three horses showing detectable prior immunity to equine influenza virus as follows. EIV-P821, produced as described in Example 1A, was grown in eggs as described in Example 2A and was formulated into a therapeutic composition comprising 10⁷ pfu EIV-P821/2ml BSA-MEM solution as described in Example 2C.

Three ponies having prior detectable hemagglutination inhibition (HAI) titers to equine influenza virus were inoculated with a therapeutic composition comprising EIV-P821 by the following method. Each pony was given a 2-ml dose of EIV-P821, administered intranasally using a syringe fitted with a blunt cannula long enough to reach past the false nostril, 1 ml per nostril.

The ponies were observed for approximately 30 minutes immediately following and at approximately four hours after vaccination for immediate type allergic reactions such as sneezing, salivation, labored or irregular breathing, shaking, anaphylaxis, or fever. The animals were further monitored on days 1-11 post-vaccination for delayed type allergic reactions, such as lethargy or anorexia. None of the three ponies in this study exhibited any allergic reactions from the vaccination.

The ponies were observed daily, at approximately the same time each day, starting two days before vaccination and continuing through day 11 following vaccination for clinical signs consistent with equine influenza. The ponies were observed for nasal discharge, ocular discharge, anorexia, disposition, heart rate, capillary refill time, respiratory rate, dyspnea, coughing, lung sounds, presence of toxic line on upper gum, and body temperature. In addition submandibular and parietal lymph nodes were palpated and any abnormalities were described. None of the three ponies in this study exhibited any abnormal reactions or overt clinical signs during the observation period.

To test for viral shedding in the animals, on days 0 through 11 following vaccination, nasopharyngeal swabs were collected from the ponies as described in Chambers, et al., 1995, Equine Practice, 17, 19-23. Chambers, et al., ibid. Briefly, two sterile Dacron polyester tipped applicators (available, e.g., from Hardwood Products Co., Guilford, Me.) were inserted, together, into each nostril of the ponies. The swabs (four total, two for each nostril) were broken off into a 15-ml conical centrifuge tube containing 2.5 ml of chilled transport medium comprising 5% glycerol, penicillin, streptomycin, neomycin, and gentamycin in PBS at physiological pH. Keeping the samples on wet ice, the swabs were aseptically wrung out into the medium and the nasopharyngeal samples were divided into two aliquots. One aliquot was used to attempt isolation of EIV by inoculation of embryonated eggs, using the method described in Example 1. The AF of the inoculated eggs was then tested for its ability to cause hemagglutination, by standard methods, indicating the presence of equine influenza virus in the AF. On days 2 and 3 post-vaccination, the other aliquots were tested for virus by the Directigen® Flu A test, available from Becton-Dickinson (Cockeysville, Md.).

Attempts to isolate, EIV from the nasopharyngeal secretions of the three animals by egg inoculation were unsuccessful. However on days 2 and 3, all animals tested positive for the presence of virus shedding using the Directigen Flu A test, consistent with the hypothesis that EIV-P821 was replicating in the seropositive ponies.

To test the antibody titers to EIV in the inoculated animals described in this example, as well as in the animals described in Examples 4-7, blood was collected from the animals prior to vaccination and on designated days post-vaccination. Serum was isolated and was treated either with trypsin/periodate or kaolin to block the nonspecific inhibitors of hemagglutination present in normal sera. Serum samples were tested for hemagglutination inhibition (HAI) titers against a recent EIV isolate by standard methods, described, for example in the Supplemental assay method for conducting the hemagglutination inhibition assay for equine influenza virus antibody (SAM 124), provided by the U.S.D.A. National Veterinary Services Laboratory under 9 CFR 113.2.

The HAI titers of the three ponies are shown in Table 5. As can be seen, regardless of the initial titer, the, serum HAI titers increased at least four-fold in all three animals after vaccination with, EIV-P821.

These data demonstrate that cold-adapted equine influenza virus EIV-P821 is safe, and non-reactogenic in sero-positive ponies, and that these animals exhibited an increase in antibody titer to equine influenza virus, even though they had prior demonstrable titers.

TABLE 5 HAI titers of vaccinated animals* [t20] Animal HAI Titer (days after vaccination) ID 0 7 14 21 18 40 80 160 160 19 10 20 40 80 25 20 40 320 80 *HAI titers are expressed as the reciprocal of the highest dilution of serum which inhibited hemagglutination of erythrocytes by a recent isolate of equine influenza virus.

EXAMPLE 4

This Example discloses an animal study to evaluate the safety and efficacy of a therapeutic composition comprising cold-adapted equine influenza virus EIV-P821.

A therapeutic composition comprising cold-adapted equine influenza virus EIV-P821 was tested for attenuation, as well as its ability to protect horses from challenge with virulent equine influenza virus, as follows. EIV-P821, produced as described in Example 1, was grown in eggs as described in Example 2A and was formulated into a therapeutic composition comprising 10⁷ pfu of virus/2 ml water, as described in Example 2C. Eight EIV-seronegative ponies were used in this study. Three of the eight ponies were vaccinated with a 2-ml dose comprising 10⁷ pfu of the EIV-P821 therapeutic composition, administered intranasally, using methods similar to those described in Example 3. One pony was given 10⁷ pfu of the EIV-P821 therapeutic composition, administered orally, by injecting 6 ml of virus into the pharynx, using a 10-ml syringe which was adapted to create a fine spray by the following method. The protruding seat for the attachment of needles was sealed off using modeling clay and its cap was left in place. About 10 holes were punched through the bottom of the syringe, i.e., surrounding the seat, using a 25-gauge needle. The syringe was placed into the interdental space and the virus was forcefully injected into the back of the mouth. The remaining four ponies were held as non-vaccinated controls.

The vaccinated ponies were observed for approximately 30 minutes immediately following and at approximately four hours after vaccination for immediate type allergic reactions, and the animals were further monitored on days 1-11 post-vaccination for delayed type allergic reactions, both as described in Example 3. None of the four vaccinated ponies in this study exhibited any abnormal reactions from the vaccination.

The ponies were observed daily, at approximately the same time each day, starting two days before virus vaccination and continuing through day 11 following vaccination for clinical signs, such as those described in Example 3. None of the four vaccinated ponies in this study exhibited any clinical signs during the observation period. This result demonstrated that cold-adapted equine influenza virus EIV-P821 exhibits the phenotype of attenuation.

To test for viral shedding in the vaccinated animals, on days 0 through 11 following vaccination, nasopharyngeal swabs were collected from the ponies as described in Example 3. The nasopharyngeal samples were tested for virus in embryonated chicken eggs according to the method described in Example 3.

As shown in Table 6, virus was isolated from only one vaccinated animal using the egg method. However, as noted in Example 3, the lack of isolation by this method does not preclude the fact that virus replication is taking place, since replication may be detected by more sensitive methods, e.g., the Directigen Flu A test.

TABLE 6 Virus isolation in eggs after vaccination. [t22] Virus Isolation (days after vaccination) ID # Route 0 1 2 3 4 5 6 7 8 9 10 11 91 IN − − + + + + + + + + + − 666 IN − − − − − − − − − − − − 673 IN − − − − − − − − − − − − 674 Oral − − − − − − − − − − − −

To test the antibody titers to equine influenza virus in the vaccinated animals, blood was collected from the animals prior to vaccination and on days 7, 14, 21, and 28 post-vaccination. Serum samples were isolated and were tested for hemagglutination inhibition (HAI) titers against a recent EIV isolate according to the methods described in Example 3.

The HAI titers of the four vaccinated ponies are shown in Table 7.

TABLE 7 HAI titers after vaccination. [t11] HAI Titer (days after vaccination) ID # Route 0 7 14 21 28 91 IN <10 <10 <10 <10 <10 666 IN 10 10 10 20 20 673 IN 10 10 10 20 20 674 Oral 20 40 40 40 40

Unlike the increase in HAI titer observed with the three animals described in the study in Example 3, the animals in this study did not exhibit a significant increase, i.e., greater than four-fold, in HAI titer following vaccination with EIV-P821.

Approximately four and one-half months after vaccine virus administration, all 8 ponies, i.e., the four that were vaccinated and the four non-vaccinated controls, were challenged by the following method. For each animal, 10⁷ pfu of the virulent equine influenza virus strain;A/equine/Kentucky/1/91 (H3N8) was suspended in 5 ml of water. A mask was connected to a nebulizer, and the mask was placed over the animal's muzzle, including the nostrils. Five (5) ml was nebulized for each animal, using settings such that it took 5-10 minutes to deliver the full 5 ml. Clinical observations, as described in Example 3, were performed on all animals three days before challenge and daily for 11 days after challenge.

Despite the fact that the vaccinated animals did not exhibit marked increases in their HAI titers to equine influenza virus, all four vaccinated animals were protected against equine influenza virus challenge. None of the vaccinated animals showed overt clinical signs or fever, although one of the animals had a minor wheeze for two days. On the other hand, all four non-vaccinated ponies shed virus and developed clinical signs and fever typical of equine influenza virus infection. Thus, this example demonstrates that a therapeutic composition of the present invention can protect horses from equine influenza disease.

EXAMPLE 5

This Example discloses an additional animal study to evaluate attenuation of a therapeutic composition comprising cold-adapted equine influenza virus EIV-P821, and its ability to protect vaccinated horses from subsequent challenge with virulent equine influenza virus. Furthermore, this study evaluated the effect of exercise stress on the safety and efficacy of the therapeutic composition.

A therapeutic composition comprising cold-adapted equine influenza virus EIV-P821 was tested for safety and efficacy in horses, as follows. EIV-P821, produced as described in Example; 1, was grown in eggs as described in Example 2A and was formulated into a therapeutic composition comprising 10⁷ pfu virus/5 ml water, as described in Example 2C. Fifteen ponies were used in this study. The ponies were randomly assigned to three groups of five animals each, as shown in Table 8, there being two vaccinated groups and one unvaccinated control group. The ponies in group 2 were exercise stressed before vaccination, while the ponies in vaccinate group 1 were held in,a stall.

TABLE 8 Vaccination/challenge protocol. [t12] Group No. Ponies Exercise Vaccine Challenge 1 5 — Day 0 Day 90 2 5 Days −4 to 0 Day 0 Day 90 3 5 — — Day 90

The ponies in group 2 were subjected to exercise stress on a treadmill prior to vaccination, as follows. The ponies were acclimated to the use of the treadmill by 6 hours of treadmill use at a walk only. The actual exercise stress involved a daily exercise regimen starting 4 days before and ending on the day of vaccination (immediately prior to vaccination). The treadmill exercise regimen is shown in Table 9.

TABLE 9 Exercise regimen for the ponies in Group 2. [t13] Speed (m/sec) Time (min.) Incline (°) 1.5 2 0 3.5 2 0 3.5 2 7 4.5† 2 7 5.5† 2 7 6.5† 2 7 7.5† 2 7 8.5† 2 7 3.5 2 7 1.5 10 0† †Speed, in meters per second (m/sec) was increased for each animal every 2 minutes until the heart rate reached and maintained ≧200 per minute

Groups 1 and 2 were given a therapeutic composition comprising 10⁷ pfu of EIV-P821, by the nebulization method described for the challenge described in Example 4. None of the vaccinated ponies in this study exhibited any immediate or delayed allergic reactions from the vaccination.

The ponies were observed daily, at approximately the same time each day, starting two days before vaccination and continuing through day 11 following vaccination for clinical signs, such as those described in Example 3. None of the vaccinated ponies in this study exhibited any overt clinical signs during the observation period.

To test for viral shedding in the vaccinated animals, before vaccination and on days 1 through 11 following vaccination, nasopharyngeal swabs were collected from the ponies as described in Example 3. The nasopharyngeal samples were tested for virus in embryonated chicken eggs according to the method described in Example 3. Virus was isolated from the vaccinated animals, i.e., Groups 1 and 2, as shown in Table 10.

TABLE 10 Virus isolation after vaccination. [t7] Virus Isolation (days after isolation) Group ID # Exercise 0 1 2 3 4 5 6 7 8 9 10 11 1 12 No − − + + + + + − + + − − 1 16 No − − + + + + + − − − − − 1 17 No − − + + + + + + + − + − 1 165 No − − − − − − − − − − − − 1 688 No − − − − − + − + − − − − 2 7 Yes − − − + + + + − − − − − 2 44 Yes − − − − − − − − − − − − 2 435 Yes − − + + + + − − − − − − 2 907 Yes − − − + − + + − − − − − 2 968 Yes − − − − − + − + − − − —

To test the antibody titers to equine influenza virus in the vaccinated animals, blood was collected prior to vaccination and on days 7, 14, 21, and 28 post-vaccination. Serum samples were isolated and were tested for HAI titers against a recent EIV isolate according to the methods described in Example 3. These titers are shown in Table 11.

TABLE 11 HAI titers after vaccination and after challenge on day 90. Animal Day Post-vaccination Group ID −1 7 14 21 28 91 105 112 119 126 1 12 <10 <10 <10 <10 <10 <10 80 320 320 640 1 16 <10 <10 20 20 <10 <10 20 160 320 320 1 17 <10 <10 10 10 10 10 80 160 160 160 1 165 <10 <10 10 10 10 10 80 80 80 80 1 688 <10 <10 20 20 20 20 20 20 20 40 2 7 <10 <10 10 10 <10 <10 20 80 80 40 2 44 <10 <10 20 20 20 10 80 320 320 320 2 435 <10 <10 20 20 10 <10 20 80 80 80 2 907 <10 <10 10 10 20 10 10 40 80 80 2 968 <10 <10 <10 <10 <10 <10 40 160 160 160 3 2 <10 80 640 640 320 3 56 <10 80 320 320 320 3 196 <10 20 160 80 80 3 198 10 40 160 320 320 3 200 <10 20 80 80 40 Group Description

On day 90 post vaccination, all 15 ponies were challenged with 10⁷ pfu of equine influenza virus strain A/equine/Kentucky/1/91 (H3N8) by the nebulizer method as described in Example 4. Clinical observations, as described in Example 3, were performed on all animals three days before challenge and daily for 11 days after challenge. There were no overt clinical signs observed in any of the vaccinated ponies. For of the five non-vaccinated ponies developed fever and clinical signs typical of equine influenza virus infection.

Thus, this, this example demonstrates that a therapeutic composition of the present invention protects horses against equine influenza disease, even if the animals are stressed prior to vaccination.

EXAMPLE 6

This Example compared the infectivities of therapeutic compositions of the present invention grown in eggs and grown in tissue culture cells. From a production standpoint, there is an advantage to growing therapeutic compositions of the present invention in tissue culture rather than in embryonated chicken eggs. Equine influenza virus, however, does not grow to as high a titer in cells as in eggs. In addition, the hemagglutinin of the virus requires an extracellular proteolytic cleavage by trypsin-like proteases for infectivity. Since serum contains trypsin inhibitors, virus grown in cell culture must be propagated in serum-free medium that contains trypsin in order to be infectious. It is well known by those skilled in the art that such conditions are less than optimal for the viability of tissue culture cells. In addition, these growth conditions may select for virus with altered binding affinity for equine cells, which may affect viral infectivity since the virus needs to bind efficiently to the animal's nasal mucosa to replicate and to stimulate immunity. Thus, the objective of the study disclosed in this example was to evaluate whether the infectivity of therapeutic compositions of the present invention was adversely affected by growth for multiple passages in in vitro tissue culture.

EIV-P821, produced as described in Example 1, was grown in eggs as described in Example 2A or in MDCK cells as described in Example 2B. In each instance, the virus was passaged five times. EIV-P821 was tested for its cold-adaptation and temperature sensitive phenotypes after each passage. The egg and cell-passaged virus preparations were formulated into therapeutic compositions comprising 10⁷ pfu virus/2 ml BSA-MEM solution, as described in Example 2C, resulting in an egg-grown EIV-P821 therapeutic composition and an MDCK cell-grown EIV-P821 therapeutic composition, respectively.

Eight ponies were used in this study. Serum from each of the animals was tested for HAI titers to equine influenza virus prior to the study. The animals were randomly assigned into one of two groups of four ponies each. Group A received the egg-grown EIV-P821 therapeutic composition, and Group B received the MDCK-grown EIV-P821 therapeutic composition, prepared as described in Example 2B. The therapeutic compositions were administered intranasally by the method described in Example 3.

The ponies were observed daily, at approximately the same time each day, starting two days before vaccination and continuing through day 11 following vaccination for allergic reactions or clinical signs as described in Example 3. No allergic reactions or overt clinical signs were observed in any of the animals.

Nasopharyngeal swabs were collected before vaccination and daily for 11 after vaccination. The presence of virus material in the nasal swabs was determined by the detection of CPE on MDCK cells infected as described in Example 1, or by inoculation into eggs and examination of the ability of the infected AF to cause hemagglutination, as described in Example 3. The material was tested for the presence of virus only, and not for titer of virus in the sample. Virus isolation results are listed in Table 12. Blood was collected and serum samples from days 0, 7, 14, 21 and 28 after vaccination were tested for agglutination inhibition antibody titer against a recent isolate. HAI titers are so in Table 12.

TABLE 12 HAI titers and virus isolation after vaccination. HAI Titer (DPV³) Virus Isolation¹ (DPV³) Group² ID 0 7 14 21 28 0 1 2 3 4 5 6 7 8 9 10 11 1 31 <10 20 160 160 160 − EC — C EC EC C C EC — — — 37 <10 40 160 160 160 — EC C C EC C C C — — — — 40 <10 20 80 160 80 — EC EC C — C EC C — EC EC — 41 <10 40 160 160 80 — EC EC C EC C EC EC — — — — 2 32 <10 <10 80 80 40 — EC — C — C — C — EC — — 34 <10 20 160 160 160 — EC — C EC C EC C — — — — 35 <10 <10 80 80 40 — EC — C — C — C — EC — — 42 <10 <10 80 80 40 — — — C — C EC EC — — — — ¹E = Egg isolation positive; C = CPE isolation positive; — = virus not detected by either of the methods ²Group 1: Virus passaged 5X in MDCK cells; Group 2: Virus passaged 5X in Eggs ³Days Post-vaccination

The results in Table 12 show that there were no significant differences in infectivity or immunogenicity between the egg-grown and MDCK-grown EIV-P821 therapeutic compositions.

EXAMPLE 7

The example evaluated the minimum dose of a therapeutic composition comprising a cold-adapted equine influenza virus required to protect a horse from equine influenza virus infection.

The animal studies disclosed in Examples 3-6 indicated that a therapeutic composition of the present invention was efficacious and safe. In those studies, a dose of 10⁷ pfu, which correlates to approximately 10⁸ TCID₅₀ units, was used. However, from the standpoints of cost and safety, it is advantageous to use the minimum virus titer that will protect a horse from disease caused by equine influenza virus. In this study, ponies were vaccinated with four different doses of a therapeutic composition comprising a cold-adapted equine influenza virus to determine the minimum dose which protects a horse against virulent equine influenza virus challenge.

EIV-P821, produced as described in Example 1A, was passaged and grown in MDCK cells as described in Example 2B and was formulated into a therapeutic composition comprising either 2×10⁴, 2×10⁵, 2×10⁶, or 2×10⁷ TCID₅₀ units/1 ml BSA-MEM solution as described in Example 2C. Nineteen horses of various ages and breeds were used for this study. The horses were assigned to four vaccine groups, one group of three horses and three groups of four horses, and one control group of four horses (see Table 13). Each of the ponies in the vaccine groups were given a 1-ml dose of the indicated therapeutic composition, administered intranasally by methods similar to those described in Example 3.

TABLE 13 Vaccination protocol. [t19] Group No. No. Animals Vaccine Dose, TCID₅₀ Units 1 3 2 × 10⁷ 2 4 2 × 10⁶ 3 4 2 × 10⁵ 4 4 2 × 10⁴ 5 4 control

The ponies were observed for approximately 30 minutes immediatelyfollowing and at approximately four hours after vaccination for immediate type reactions, and the animals were further monitored on days 1-11 post-vaccination for delayed type reactions, both as described in Example 3. None of the vaccinated ponies in this study exhibited any abnormal reactions or overt clinical signs from the vaccination.

Blood for serum analysis was collected 3 days before vaccination, on days 7, 14, 21, and 28 after vaccination, and after challenge on Days 35 and 42. Serum samples were tested for HAI titers against a recent EIV isolate according to the methods described in Example 3. These titers are shown in Table 14. Prior to challenge on day 29, 2 of the 3 animals in group 1, 4 of the 4 animals in group 2, 3 of the 4 animals in group 3, and 2 of the 4 animals in group 4 showed at least 4-fold increases in HAI titers after vaccination In addition, 2 of the 4 control horses also exhibited increases in HAI titers. One interpretation for this result is that the control horses were exposed to vaccine virus transmitted from the vaccinated horses, since all the horses in this study were housed in the same barn.

TABLE 14: HAI titers post-vaccination and post-challenge, and challenge results.

TABLE 14 HAI titers post-vaccination and post-challenge, and challenge results. Vaccination on Day 0, Dose in Animal Challenge on Day 29 Chall. Sick No. TCID₅₀ units ID −1 7 14 21 28 35 42 +/− 1 2 × 10⁷ 41 <10 <10 10 40 10 20 80 − 42 40 40 40 40 40 <10 80 − 200 <10 <10 80 40 160 40 40 − 2 2 × 10⁶ 679 <10 10 40 40 40 20 20 − 682 <10 <10 40 40 40 40 40 − 795 20 80 160 160 320 320 640 − R <10 10 40 20 160 40 40 − 3 2 × 10⁵ 73 <10 <10 160 40 80 160 160 − 712 <10 <10 20 20 40 40 20 − 720 <10 20 80 40 80 80 160 − 796 <10 <10 <10 <10 <10 10 80 + 4 2 × 10⁴ 75 <10 <10 <10 <10 <10 <10 160 + 724 <10 >10 <10 <10 <10 20 320 + 789 <10 10 320 160 320 320 320 − 790 <10 <10 80 40 160 80 40 5 Control 12 <10 <10 <10 20 20 40 40 − 22 10 20 40 10 160 40 640 − 71 <10 <10 <10 <10 10 20 160 + 74 <10 <10 <10 <10 <10 <10 20 +

On day 29 post vaccination, all 19 ponies were challenged with equine influenza virus strain A/equine/Kentucky/1/91 (H3N8) by the nebulizer method as described in Example 4. The challenge dose was prospectively calculated to contain about 10⁸ TCID₅₀ units of challenge virus in a volume of 5 ml for each animal. Clinical observations, as described in Example 3, were monitored beginning two days before challenge, the day of challenge, and for 11 days following challenge. As shown in Table 14, no animals in groups 1 or 2 exhibited clinical signs indicative of equine influenza disease, and only one out of four animals in group 3 became sick. Two out of four animals in group 4 became sick, and only two of the four control animals became sick. The results in Table 14 suggest a correlation between seroconversion and protection from disease, since, for example, the two control animals showing increased HAI titers during the vaccination period did not show clinical signs of equine influenza disease following challenge. Another interpretation, however, was that the actual titer of the challenge virus may have been less than the calculated amount of 10⁸ TCID₅₀ units, since, based on prior results, this level of challenge should have caused disease in all the control animals.

Nonetheless, the levels of seroconversion and the lack of clinical signs in the groups that received a therapeutic composition comprising at least 2×10⁶ TCID₅₀ units of a cold-adapted equine influenza virus suggests that this amount was sufficient to protect a horse against equine influenza disease. Furthermore, a dose of 2×10⁵ TCID₅₀ unit induced seroconversion and gave clinical protection from challenge in 3 out of 4 horses, and thus even this amount may be sufficient to confer significant protection in horses against equine influenza disease.

EXAMPLE 8

This example discloses an animal study to evaluate the duration of immunity of a therapeutic composition comprising cold-adapted equine influenza virus EIV-P821.

A therapeutic composition comprising cold-adapted equine influenza virus EIV-P821, produced as described in Example 1, was grown in eggs similarly to the procedure described in Example 2A, was expanded by passage in MDCK cells similarly to the procedure described in Example 2B, and was formulated into a therapeutic composition as described in Example 2C. Thirty horses approximately 11 to 12 months of age were used for this study. Nineteen of the horses were each vaccinated intranasally into one nostril using a syringe with a delivery device tip attached to the end, with a 1.0 ml dose comprising 6 logs of TCID₅₀ units of the EIV-P821 therapeutic composition. Vaccinations were performed on Day 0.

The horses were observed on Day 0 (before vaccination and up to 4 hours post-vaccination) and on Study Days 1, 2, 3, 7, 15, and 169 post-vaccination. On these days, a distant examination for a period of at least 15 minutes was performed. This distant examination included observation for demeanor, behavior, coughing, sneezing, and nasal discharge. The examination on Day 169 also served to confirm that the horses were a condition of health suitable for transport to the challenge site which was located approximately 360 miles from the vaccination site.

The animals were acclimated to the challenge site and were observed approximately daily by a veterinarian or animal technician for evidence of disease. A general physical examination was performed on Day 171 post-vaccination to monitor the following: demeanor, behavior, coughing, sneezing, and nasal discharge. From Days 172 to 177, similar observations as well as rectal temperature were recorded, according to the judgment of the attending veterinarian for any individual horse with abnormal clinical presentation.

No vaccinated horses showed any adverse reactions post-vaccination. One vaccinate was found dead about two months after vaccination. This horse showed no evidence of adverse reaction when observed for at least one month after vaccination. Although no cause oft death could be firmly established, the death was not instantaneous and was considered to be consistent with possible contributing factors such as colic, bone fracture, or severe worm burden. Since there was no other evidence for any adverse reactions post-vaccination in any other vaccinates, it is highly unlikely that the vaccine contributed to any adverse reaction in this case.

Challenges were performed on Day 181 post-vaccination. The following wild-type isolate of equine influenza virus previously shown to cause disease in horses was used as the challenge virus: A/equine/2/Kentucky/91. Prior to infection of each challenge group, the challenge; material was rapidly thawed at approximately 37° C. The virus was diluted with phosphate-buffered saline to a total volume of approximately 21 ml. The diluted material was stored chilled on ice until immediately before inoculation. Before inoculation and at the end of nebulization for each challenge group, a sample of diluted challenge virus was collected for pre- and post-inoculation virus titer confirmation. Vaccinates and controls were randomly assigned to 4 challenge groups of 6 horses each and, one challenge group of 5 horses so that each challenge group contained a mixture of 4 vaccinates and 2 controls or 3 vaccinates and 2 controls.

Challenge virus in aerosol form was delivered through a tube inserted through a small opening centrality in the plastic ceiling with an ultrasonic nebulizer (e.g., DeVilbiss Model 099HD, DeVilbiss Healthcare Inc., Somerset, Pa.) for a period of approximately 10 minutes. The horses remained in the chamber for a further period of appiroximately 30 minutes after the nebulization had been completed (total exposure time, approximately 40 minutes). At that time, the plastic was removed to vent the chamber, and the horses were released and returned to their pen. The challenge procedure was repeated for each group.

All statistical methods in this study were performed using SAS (SAS Institute, Cary, N.C. and P<0.05 was considered to be statistically significant. Beginning on Day 178 post-vaccination (three days prior to challenge) through Day 191 (day 10 postthe horses were observed daily by both distant and individual examinations. Rectal temperatures were measured at these times. Data from day 0 (challenge day) to day 10 were included in the analysis; see Table 15.

TABLE 15 Effect of challenge on daily temperatures (E C) in vaccinated and controlhorses (least squares means). [t9] Day post Vaccinated non-vaccinated challenge (n = 19) (n = 10) P-value 0 100.7 100.8 0.8434 1 100.5 100.4 0.7934 2 103.4 104.9 0.0024 3 101.8 103.9 0.0001 4 101.5 103.2 0.0002 5 101.7 103.8 0.0001 6 101.3 103.6 0.0001 7 100.7 102.3 0.0007 8 100.5 101.4 0.0379 9 100.1 100.3 0.7416 10  100.3 100.5 0.7416 pooled SEM* 0.27 0.38 *Standard error of the mean.

Table 15 shows that on days 2 through 8, vaccinated horses had lower temperatures (P<0.05 than the non-vaccinated control horses.

The distant examination consisted of a period of 20 minutes where the following observations were made: coughing, nasal discharge, respiration, and depression. Scoring criteria are shown in Table 16.

TABLE 16 Clinical signs and scoring index. Clinical Sign Description Score Coughing normal during observation period of 15 min 0 coughing once during observation 1 coughing twice or more during observation 2 Nasal discharge normal 0 abnormal, serous 1 abnormal, mucopurulent 2 abnormal, profuse 3 Respiration normal 0 abnormal (dyspnea, tachypnea) 1 Depression normal 0 depression present^(†) 1 ^(†)Depression was assessed by subjective evaluation of individual animal behavior that included the following: failure to approach food rapidly, general lethargy, inappetence, and anorexia.

Each horse was scored for each of these categories. Additionally, submandibular lymph nodes were palpated to monitor for possible bacterial infection. In any case where there was a different value recorded for a subjective clinical sign score from an observation on the same day at the distant versus the individual examination, the greater score was used in the compilation and analysis of results. For purposes of assessing the health of the horses prior to final disposition, distant examinations were performed at 14, 18, and 21 days post-challenge. Data from days 1 through 10 post-challenge were included in the analysis. These scores were summed on each day for each horse, and the vaccinates and controls were compared using the Wilcoxon rank sums test. In addition, these scores were summed across all days for each horse, and compared in the same manner. The mean ranks and mean clinical scores are shown in Tables 17 and 18, respectively. Five days post-challenge, the mean rank of scores in the vaccinated horses was lower (P<0.05) than in the non-vaccinated control horses; and this effect continued on days 6, 7, 8, 9, and 10 (P<0.05). The cumulative rank over the entire test period was also lower (P<0.05) in the vaccinated horses than the noncontrols.

TABLE 17 Effect of challenge on clinical sign scores in vaccinated and control horses (mean rank). [t4] Vaccinated Non-vaccinated Day post (n = 19), (n = 10), challenge mean rank* mean rank P-value 0 13.6 17.6 0.1853 1 16.4 12.4 0.2015 2 15.1 14.9 0.9812 3 13.3 18.3 0.1331 4 13.5 17.9 0.1721 5 12.4 19.9 0.0237 6 12.7 19.4 0.0425 7 12.1 20.6 0.0074 8 12.6 19.6 0.0312 9 13.1 18.7 0.0729 10  12.3 20.1 0.0135 total over 11 11.8 21.2 0.0051 days *By Wilcoxon rank sum test.

horses (mean scores).

Day post Vaccinated Non-vaccinated challenge (n = 19) (n = 10) 0 1.2 1.6 1 1.5 0.9 2 2.4 2.5 3 3.2 4.1 4 3.4 4.3 5 3.2 4.7 6 3.4 4.8 7 3.3 4.7 8 3.2 4.5 9 3.2 3.9 10 2.4 3.4

Nasopharyngeal swabs were obtained on the day prior to challenge and on days 1 to 8 post-challenge, as described in Example 3, and tested for shed virus by cell culture assay. The percent of horses shedding challenge virus in each group is shown in Table 19. The percent of horses shedding the challenge virus in the vaccinated group was lower (P<0.05) on days 5 and 6 post-challenge than in the non-vaccinated controls. The mean number of days the challenge virus was shed was also lower (P<0.05) in the vaccinated group as compared to the non-vaccinated controls.

TABLE 19 Percent of horses shedding virus per day post-challenge and mean number of days of shedding per group. [t18] Day post Vaccinated Non-vaccinated challenge (n = 19) (n = 10) −1   0 0 1 63.2 90 2 100 100 3 84.2 100 4 100 100 5 47.4 88.9* 6 10.5 77.8* 7 5.3 20 8 0 0 average number of days 4.1 5.6* shedding *Within a time point, vaccinates different from non-vaccinates (P0.05) by either Fisher”s exact test (percent data) or Wilcoxon rank sums test (days shedding).

The scores from clinical signs relevant to influenza and the objective temperature measurements both demonstrated a statistically significant reduction in the group of vaccinates when compared to those from the control group; this is consistent with an interpretation that the vaccine conferred significant protection from disease.

The ability of horses to shed influenza virus post-challenge was also significantly reduced in vaccinates as compared to controls in both the incidence of horses positive for shedding on certain days post-challenge and the mean number of days of shedding per horse. This decreased shedding by vaccinates is important in that it should serve to reduce the potential for exposure of susceptible animals to the wild-type virus in an outbreak of influenza.

The results of this study are consistent with the interpretation that the vaccine safely conferred protection for 6 months from clinical disease caused by equine influenza and reduced the potential for the spread of naturally occurring virulent equine influenza virus. While the degree of protection from disease was not complete (13 out of 19 vaccinates were protected, while 10/10 controls were sick), there was a clear reduction in the severity and duration of clinical illness and a noticeable effect on the potential for viral shedding after exposure to a virulent strain of equine influenza. The finding that both vaccinates and controls were seronegative immediately prior to challenge at 6 months post-immunization suggests that immunity mediated by something other than serum antibody may be of primary importance in the ability of this vaccine to confer measurable and durable protection.

EXAMPLE 9

This Example discloses an animal study to evaluate the ability of a therapeutic composition comprising cold-adapted equine influenza virus EIV-P821 to aid in the prevention of disease following exposure to a heterologous strain of equine influenza virus.

The heterologous strain tested was A/equine/2/Saskatoon/90, described genetically as a Eurasian strain (obtained from Hugh Townsend, University of Saskatchewan). Twenty female Percheron horses approximately 15 months of age (at the time of vaccination) were used for the efficacy study. The horses were assigned to two groups, one group of 10 to be vaccinated and another group of 10 to serve as non-vaccinated controls. On day 0, the vaccinate group was vaccinated in the manner described in Example 8.

The challenge material, i.e. equine flu strain A/equine/2/Saskatoon/90 [H3N8] was prepared similarly to the preparation in Example 8. Vaccinates and controls were randomly assigned to 4 challenge groups of 5 horses each such that each challenge group contained a mixture of 2 vaccinates and three controls or vice versa. The challenge procedure was similar to that described in Example 8. Challenges were performed on Day 28 post-vaccination.

Clinical observations were performed for the vaccinates and controls on Day -4 and on Study Days 0 (before vaccination and up to 4 hours post-vaccination), 1 to 7, 12, 15 to 17, 19 to 23, 25 to 38, and 42. For days on which clinical observations were performed during Days -4 to 42, clinical observations including rectal temperature were recorded according to the judgment of the attending veterinarian for any individual horse with abnormal clinical presentation. Horses were scored using the same criteria as in Example 8 (Table 15). Distant examinations were performed on these days as described in Example 8. On Day 20 and from Days 25 to 38, the horses were also observed by both distant and individual examinations (also performed as described in Example 8).

Rectal temperatures were measured daily beginning 3 days prior to challenge, and continuing until 10 days post-challenge. Day 0 is the day relative to challenge. Data from days 0 through 10 were included in the analysis. Statistical methods and criteria were identical to those used in Example 8. On days 2, 5 and 7, vaccinated horses had statistically significant lower body temperatures than the non-vaccinated control horses (Table 20).

TABLE 20 Effect of challenge on daily temperatures (E C) in vaccinated and control horses (least squares means). [t5] Day post Vaccinated Non-vaccinated challenge (n = 10) (n = 10) P-value 0 99.9 99.8 0.9098 1 100.5 100.3 0.4282 2 101.0 102.8 0.0001 3 100.7 100.6 0.7554 4 101.0 101.3 0.4119 5 100.8 102.1 0.0004 6 100.4 100.4 0.9774 7 100.3 101.1 0.0325 8 100.6 100.7 0.8651 9 100.5 100.6 0.8874 10 100.5 100.1 0.2465 Standard error of the mean = 0.249.

Data from days 1 through 10 post-challenge were included in the analysis. These scores were summed on each day for each horse, and the vaccinates and controls were compared using the Wilcoxon rank sums test. All statistical methods were performed as described in Example 9. In addition, these scores were summed across all days for each horse, and compared in the same manner. Mean ranks are shown in Table 21.

TABLE 21 Effect of challenge on clinical sign scores in vaccinated and control horses (mean rank). [t6] Day post Vaccinated Non-vaccinated challenge (n = 10) (n = 10) P-value* 1 8.85 12.15 0.1741 2 8.80 12.20 0.1932 3 8.90 12.10 0.2027 4 7.60 13.40 0.0225 5 6.90 14.10 0.0053 6 7.00 14.00 0.0059 7 6.90 14.10 0.0053 8 7.60 13.40 0.0251 9 6.90 14.10 0.0048 10  6.10 14.90 0.0006 total over 10 days 5.70 15.30 0.0003 *By Wilcoxon 2 sample test.

On day 4 post-challenge, the mean rank of scores in the vaccinated horses was lower (P<0.05) than the non-vaccinated control horses, and this effect continued throughout the remainder of the study (P<0.05). The cumulative rank over the entire test period was also lower in the vaccinated horses than the noncontrols (P<0.05).

Nasopharyngeal:swabs were collected on days 1 and 8 post-challenge, as described in Example 3. The nasal samples were analyzed for the presence of virus by cell inoculation with virus detection by cytopathogenic effect (CPE) or by egg inoculation with virus detection by hemagglutination (HA). The cell-culture assay was performed as generally described by Youngner et al, 1994, J. Clin. Microbiol. 32, 750-754. Serially diluted nasal samples were added to wells containing monolayers of Madin Darby Canine Kidney (MDCK) cells. After incubation, wells were examined for the presence and degree of cytopathogenic effect. The quantity of virus in TCID₅₀ units was calculated by the Reed-Muench technique. The egg infectivity assay was performed as described in Example 1. The percent of horses shedding challenge virus for each assay in each group is shown in Tables 22 and 23. The percent of horses shedding the challenge virus in the vaccinated group was lower (P<0.05) on days 2 through 7 post-challenge by either method. No differences were seen on days 1 or 8 post-challenge. The number of days the challenge virus was shed was also lower (P<0.05) in the vaccinated group as compared to the non-vaccinated controls; see Tables 22 and 23.

TABLE 22 Percent of horses shedding virus following challenge- cell culture assay. [t15] Day post Vaccinated Non-vaccinated challenge (n = 10) (n = 10) 1 0  0 2 0  70* 3 0  70* 4 20 100* 5 10 100* 6 20 100* 7 0  80* 8 0  30 average number of days 0.5  5.5* shedding *Within a time point, vaccinates different from non-vaccinates, P < 0.05 by either Fisher's exact test (percent data) or Wilcoxon 2 sample test (days shedding)

TABLE 23 Percent of horses shedding virus following challenge- egg infectivity assay. [t16] Day post Vaccinated Non-vaccinated challenge (n = 10) (n = 10) 1 0  0 2 0 70* 3 10 70* 4 0 90* 5 10 70* 6 20 90* 7 0 50* 8 0  0 average number of days 0.4  4.4* shedding *Within a time point, vaccinates different from non-vaccinates, P < 0.05 by either Fisher”s exact test (percent data) or Wilcoxon 2 sample test (days shedding).

The extent (severity and duration) of clinical signs of influenza among vaccinates was substantially reduced relative to the controls. The scores from clinical signs relevant to influenza and the objective temperature measurements both demonstrated a statistically significant reduction in the group of vaccinates when compared to those from the control group; indicating that the vaccine conferred significant protection from disease by the heterologous strain.

The ability of horses to shed influenza virus post-challenge was also significantly reduced in vaccinates as opposed to controls in both the incidence of horses positive for shedding on certain days post-challenge and the mean number of days of shedding per horse. This decreased shedding by vaccinates is important in that it should serve to reduce the potential for exposure of susceptible animals to the wild-type virus in an outbreak of influenza.

Overall, the results of this study show that the vaccine conferred protection against a heterologous challenge by a member of the Eurasian lineage of equine influenza virus strains.

EXAMPLE 10

This Example discloses an animal study to evaluate the ability of a therapeutic composition comprising cold-adapted equine influenza virus EIV-P821 to aid in the prevention of disease following exposure to a heterologous strain of equine influenza virus.

The heterologous strain tested was A/equine/2/Kentucky/98 [H3N8](obtained from Tom Chambers, University of Kentucky). Eight ponies aged 5 to 7 months were used for this efficacy study. The horses were assigned to two groups, one group of 4 to be vaccinated and another group of 4 to serve as non-vaccinated controls. Ponies were vaccinated as described in Example 8, on Day 0.

Clinical observations were performed for the vaccinates on Study Day 0 (before vaccination and at 4 hours post-vaccination), as well as on Days 1 to 8, 23, 30 to 50, and 57 post-vaccination. Controls were observed clinically on Days 29 to 50 and 57. The observations were performed and scored as described in Example 8.

The challenge material i.e. equine flu strain from Kentucky/98, was prepared by passing the isolated virus two times in eggs. The inoculum for each horse was prepared by thawing 0.5 ml of the virus, then diluting in 4.5 ml of sterile phosphate-buffered saline. The inoculum was administered by nebulization using a mask for each individual horse on Day 36 post-vaccination.

The clinical observation scores were summed on each day for each horse, and horses were ranked according to the cumulative total score from days 1 to 9 post-challenge. Theses results are shown in Table 24.

TABLE 24 Clinical sign observations: total scores, ranked by total score. [t2] Group Halter Identity Total Score^(#) Days 1 to 9 post-challenge 1-Vaccinate 50 0 1-Vaccinate 52 0 1-Vaccinate 55 1 1-Vaccinate 15 2 2-Control 61 21 2-Control 20 25 2-Control 7 26 2-Control 13 26 ^(#)Total scores represent the sum of daily scores (where daily scores equal the sum of scores for coughing, nasal discharge, respiration, and depression) and are ranked from the lowest (least severe) to highest (most severe) scores.

The results of Table 24 show that the scores for vaccinates were between 0 and 2, which was significantly lower than the score for controls, which were between 21 and 26.

Rectal temperatures were measured daily beginning 6 days prior to challenge, and continuing until 9 days post-challenge. Day 0 is the day relative to challenge. Data from days 0 through 9 were included in the analysis. These results are shown in Table 25.

TABLE 25 Effect of challenge on daily mean temperatures (E C) in vaccinated and control horses. [t14] Day post challenge control vaccinate difference 0 99.7 99.5 0.2 1 100.0 99.6 0.4 2 103.9 100.2 3.7 3 99.8 99.2 0.6 4 99.6 99.1 0.5 5 99.8 99.3 0.5 6 99.6 99.3 0.3 7 99.3 99.0 0.3 8 99.7 99.6 0.1 9 99.5 99.1 0.4

The temperatures of the control horses were higher than the temperatures of the vaccinated horses on all days. The temperature in control horses was significantly higher on day 2.

Nasopharyngeal swabs were collected on days 1 and 8, post-challenge, as described in Example 3. These samples were tested for shed virus by an egg infectivity assay as described in Example 1. The results of the assay are shown in Table 26.

TABLE 26 Virus shedding post-challenge detected by egg infectivity. Study day 35 37 38 39 40 41 42 43 44 Days −1 1 2 3 4 5 6 7 8 post-challenge No. days Identity positive Group No. Detection of virus* per horse Vacci- 15 0 2 0 3 3 0 2 1 0 5 nates 50 0 0 0 0 0 1 0 0 0 1 52 0 0 3 3 2 2 0 0 0 4 55 0 2 3 1 3 0 0 0 0 4 No. horses 0 2 2 3 3 2 1 1 0 positive per day Con- 07 0 3 3 3 3 3 3 1 0 7 trols 13 0 3 3 3 3 3 3 1 0 7 20 0 2 3 3 3 3 3 1 0 7 61 0 3 3 3 3 3 3 2 0 7 No. horses 0 4 4 4 4 4 4 4 0 positive per day *Values refer to the number of eggs testing positive of 3 eggs tested per sample. For statistical analysis, a sample was considered positive for virus if at least 1 egg was positive per sample.

The results of Table 26 show that the number of horses positive per day was higher for the controls than for the vaccinates. Additionally, control horses were positive for more days than vaccinates.

The scores from clinical signs relevant to influenza and the objective temperature measurements both:demonstrated significant differences in the group of vaccinates when compared to the control group; this shows that the vaccine conferred significant protection from disease caused by the heterologous strain Kentucky/98.

The ability of horses to shed influenza virus post-challenge was also significantly reduced in vaccinates as opposed to controls in the mean number of days of shedding per horse. This decreased shedding by vaccinates is important in that it should serve to reduce the potential for exposure of susceptible animals to the wild-type virus in an outbreak of influenza.

Overall, the results of this study show that the vaccine safely conferred protection to a heterologous challenge by a recent and clinically relevant isolate. When the results of this study are viewed in the light of the protection previously demonstrated against heterologous challenge with a Eurasian strain (Example 9), there is clear evidence to support the assertion that this modified live vaccine can confer protection against heterologous as well as homologous equine influenza infection.

EXAMPLE 11

This example describes the cloning and sequencing of equine influenza M (matrix) protein nucleic acid molecules for wild type and cold-adapted equine influenza viruses.

A. Nucleic acid molecules encoding wild type or cold-adapted equine influenza virus M protein, were produced as follows. A PCR product containing an equine M gene was produced by PCR amplification from equine influenza virus DNA, and primers w584 and w585, designated SEQ ID NO:26, and SEQ ID NO:27, respectively. A nucleic acid molecule of 1023 nucleotides, denoted nei_(wt)M₁₀₂₃, with a coding strand having a nucleic acid sequence designated SEQ ID NO:1 was produced by further PCR amplification using the above described PCR product as a template, and cloned into pCR 2.1® TA cloning vector, available from Invitrogen, Carlsbad, Calif., using standard procedures recommended by the manufacturer. The primers used were the T7 primer,designated by SEQ ID NO:29 and the REV primer, designated by SEQ ID NO:28. Plasmid DNA was purified using a mini-prep method available from Qiagen, Valencia, Calif., PCR products were prepared for sequencing using a Prism™ Dye Terminator Cyle Sequencing Ready Reaction kit, a Prism™ dRhodamine Terminator Cycle Sequencing Ready Reaction kit, or a Prism™ BigDye™ Terminator Cycle Sequencing Ready Reaction kit, all available from PE Applied Biosystems, Foster City, Calif., following the manufacturer's protocol. Specific PCR conditions used with the kit were a rapid ramp to 95 E C, hold for 10 seconds followed by a rapid ramp to 50 E C with a 5 second hold then a rapid ramp to 60 E C with a 4 minute hold, repeating for 25 cycles. Different sets of primers were used in different reactions: T7 and REV were used in one reaction; w584 and w585 were used in a second reaction; and efM-a1, designated SEQ ID NO:31 and efM-s1, designated SEQ ID NO:30 were used in a third reaction. PCR products were purified by ethanol/magnesium chloride precipitation. Automaied sequencing of DNA samples was performed using an ABI Prism™ Model 377 with XL upgrade DNA Sequencer, available from PE Applied Biosystems.

Translation of SEQ ID NO:1 indicates that nucleic acid molecule nei_(wt)M₁₀₂₃ encodes a full-length equine influenza M protein of about 252 amino acids, referred to herein as Pei_(wt)M₂₅₂, having amino acid sequence SEQ ID NO:2, assuming an open reading frame in which the initiation codon spans from nucleotide 25 through nucleotide 28 of SEQ ID NO:1 and the termination codon spans from nucleotide 781 through nucleotide 783 of SEQ ID NO:1. The region encoding Pei_(wt)M₂₅₂, designated nei_(wt)M756, and having a coding strand comprising nucleotides 25 to 780 of SEQ ID NO:1, is represented by SEQ ID NO:3.

SEQ ID NO:1 and SEQ ID NO:3 represent the consensus sequence obtained from two wild type nucleic acid molecules, which differ in one nucleotide. Nucleotide 663 of nei_(wt1)M₁₀₂₃, i.e., nucleotide 649 of nei_(wt2)M₇₅₆, was adenine, while nucleotide 663 of nei_(wt2)M₁₀₂₃, i.e., nucleotide 649 of nei_(wt2)M₇₅₆, was guanine. Translation of these sequences does not result in an amino acid change at the corresponding amino acid; both translate to valine at residue 221 in Pei_(wt)M₂₅₂.

B. A nucleic acid molecule of 1023 nucleotides encoding a cold-adapted equine influenza virus M, denoted nei_(ca1)M₁₀₂₃, with a coding strand having a sequence designated SEQ ID NO:4 was produced by further PCR amplification and cloned into the pCR®-Blunt cloning vector available from Invitrogen, using conditions recommended by the manufacturer, and primers T7 and REV. Plasmid DNA purification and cycle sequencing were performed as described in Example 11, part A. Translation of SEQ ID NO:4 indicates that nucleic acid molecule nei_(ca1)M₁₀₂₃ encodes a full-length equine influenza M protein of about 252 amino acids, referred to herein as Pei_(ca1)M₂₅₂, having amino acid sequence SEQ ID NO:5, assuming an open reading frame in which the initiation codon spans from nucleotide 25 through nucleotide 28 of SEQ ID NO:4 and the termination codon spans from nucleotide 781 through nucleotide 783 of SEQ ID NO:4. The region encoding Pei_(ca1)M₂₅₂, designated nei_(ca1)M₇₅₆ and having a coding strand comprising nucleotides 25 to 780 of SEQ ID NO:4, is represented by SEQ ID NO:6. PCR amplification of a second nucleic acid molecule encoding a cold-adapted equine influenza M protein in the same manner resulted in molecules nei_(ca2)M₁₀₂₃, identical to nei_(ca1)M₁₀₂₃, and nei_(ca2)M₇₅₆, identical to nei_(ca1)M₇₅₆.

C. Comparison of the nucleic acid sequences of the coding strands of nei_(wt)M₁₀₂₃ (SEQ ID NO:1) and nei_(ca1)M₁₀₂₃ (SEQ ID NO:4) by DNA alignment reveals the following differences: a G to T shift at base 67, a C to T shift at base 527, and a G to C shift at base 886. Comparison of the amino acid sequences of proteins Pei_(wt)M₂₅₂ (SEQ ID NO:2) and Pei_(ca1)M₂₅₂ (SEQ ID NO:5) reveals the following differences: a V to L shift at amino acid 23 relating to the G to T shift at base 67 in the DNA sequences; and a T to I shift at amino acid 187 relating to the C to T shift at base 527 in the DNA sequences.

EXAMPLE 12

This example describes the cloning and sequencing of equine influenza HA (hemagglutinin) protein nucleic acid molecules for wild type or cold-adapted equine influenza viruses.

A. Nucleic acid molecules encoding wild type or cold-adapted equine influenza virus HA proteins were produced as follows. A PCR product containing an equine HA gene was produced by PCR amplification from equine influenza virus DNA and primers w578 and w579, designated SEQ ID NO:32 and SEQ ID NO:33, respectively. A nucleic acid molecule of 1762 nucleotides encoding a wild-type HA protein, denoted nei_(wt)HA₁₇₆₂ with a coding strand having a nucleic acid sequence designated SEQ ID NO:7 was produced by further PCR amplification using the above-described PCR product as a template and cloned into pCR 2.1® TA cloning vector as described in Example 11A. Plasmid DNA was purified and sequenced as in Example 11A, except that primers used in the sequencing kits were either T7 and REV in one case, or HA-1, designated SEQ ID NO:34, and HA-2, designated SEQ ID NO:35, in a second case.

Translation of SEQ ID NO:7 indicates that nucleic acid molecule nei_(wt)HA₁₇₆₂encodes a full-length equine influenza HA protein of about 565 amino acids, referred to herein as Pei_(wt)HA₅₆₅, having amino acid sequence SEQ ID NO:8, assuming an open reading frame in which the initiation codon spans from nucleotide 30 through nucleotide 33 of SEQ ID NO:7 and the termination codon spans from nucleotide 1725 through nucleotide 1727 of SEQ ID NO:7. The region encoding Pei_(wt)HA₅₆₅, designated nei_(wt)HA₁₆₉₅, and having a coding strand comprising nucleotides 30 to 1724 of SEQ ID NO:7 is represented by SEQ ID NO:9.

B. A nucleic acid molecule of 1762 nucleotides encoding a cold-adapted equine influenza virus HA protein, denoted nei_(ca1)HA₁₇₆₂, with a coding strand having a sequence designated, SEQ ID NO:10 was produced as described in Example 11B. Plasmid DNA purification and cycle sequencing were performed as described in Example 12, part A.

Translation of SEQ ID NO:10 indicates that nucleic acid molecule nei_(ca1)HA₁₇₆₂ encodes a full-length equine influenza HA protein of about 565 amino acids, referred to herein as Pei_(ca1)HA₅₆₅, having amino acid sequence SEQ ID NO:11, assuming an open reading frame in which the initiation codon spans from nucleotide 30 through nucleotide 33 of SEQ ID NO:10 and the termination codon spans from nucleotide 1725 through nucleotide 1727 of SEQ ID NO:10. The region encoding Pei_(ca1)HA565, designated nei_(ca1)HA₁₆₉₅, and having a coding strand comprising nucleotides 30 to 1724 of SEQ ID NO:10, is represented by SEQ ID NO:12.

PCR amplification of a second nucleic acid molecule encoding a cold-adapted equine influenza HA protein in the same manner resulted in molecules nei_(ca2)HA₁₇₆₂, identical to nei_(ca1)HA₁₇₆₂, and nei_(ca2)HA₁₆₉₅, identical to nei_(ca1)HA₁₆₉₅.

C. Comparison of the nucleic acid sequences of the coding strands of nei_(wt)HA₁₇₆₂ (SEQ ID NO:7) and nei_(ca1)HA₁₇₆₂ (SEQ ID NO:10) by DNA alignment reveals the following differences: a C to T shift at base 55, a G to A shift at base 499, a G to A shift at base 671, a C to T shift at base 738, a T to C shift at base 805, a G to A shift at base 1289, and an A to G shift at base 1368. Comparison of the amino, acid sequences of proteins Pei_(wt)HA₅₆₅ (SEQ ID NO:8) and Pei_(ca1)HA₅₆₅ (SEQ ID NO:11) reveals the following differences: a P to L shift at amino acid 18 relating to the C to T shift at base 55 in the DNA sequences; a G to E shift at amino acid 166 relating to the G to A shift at base 499 in the DNA sequences; an R to W shift at amino acid 246 relating to the C to T shift at base 738 in the DNA sequences; an M to T shift at amino acid 268 relating to the T to C shift at base 805 in the DNA sequences; a K to E shift at amino acid 456 relating to the A to G shift at base 1368 in the DNA sequences. There is no change of the serine (S) at residue 223 relating to the G to A shift at base 671 in the DNA sequences, nor is there a change of the arginine (R) at residue 429 relating to the G to A shift at base 1289 in the DNA sequences.

EXAMPLE 13

This example describes the cloning and sequencing of equine influenza PB2 protein (RNARNA polymerase) nucleic acid molecules corresponding to the N-terminal portion of the protein, for wild type or cold-adapted equine influenza viruses.

A. Nucleic acid molecules encoding wild type or cold-adapted equine influenza virus PB2-N proteins were produced as follows. A PCR product containing an N-terminal portion of the equine PB2 gene was produced by PCR amplification from equine influenza virus DNA, and primers w570 and w571, designated SEQ ID NO:36 and SEQ ID NO:37, respectively. A nucleic acid molecule of 1241 nucleotides encoding a wild type PB2-N protein, denoted nei_(wt)PB2-N₁₂₄₁, with a coding strand having a nucleic acid sequence designated SEQ ID NO:13 was produced by further PCR amplification using the above described PCR product as a template and cloned as described in Example 11B. Plasmid DNA was purified and sequenced as in Example 11B, except that only T7 and REV primers were used in the sequencing kits.

Translation of SEQ ID NO:13 indicates that nucleic acid molecule nei_(wt)PB2-N₁₂₄₁ encodes an N-terminal portion of influenza PB2 protein of about 404 amino acids, referred to herein as P_(wt)PB2-N₄₀₄, having amino acid sequence SEQ ID NO:14, assuming an, open reading frame in which the initiation codon spans from nucleotide 28 through nucleotide 30 of SEQ ID NO:13, and the last codon spans from nucleotide 1237 through nucleotide 1239. The region encoding P_(wt)PB2-N₄₀₄, designated nei_(wt)PB2-N₁₂₁₄, and having a coding strand comprising nucleotides 28 to 1239 of SEQ ID NO:13 is represented by SEQ ID NO:15.

B. A nucleic acid molecule of 1239 nucleotides encoding an N-terminal portion of influenza PB2 cold-adapted equine influenza virus PB2-N protein, denoted nei_(ca1)PB2-N₁₂₄₁, with a coding strand having a sequence designated SEQ ID NO:16 was produced, and sequenced as described in as in Example 12, part A.

Translation of SEQ ID NO:16 indicates that nucleic acid molecule nei_(ca1)PB2-N₁₂₄₁ encodes an N-terminal portion of equine influenza PB-2 protein of about 404 amino acids, referred to herein as P_(ca1)PB2-N₄₀₄, having amino acid sequence SEQ ID NO:17, assuming an open reading frame in which the initiation codon spans from nucleotide 28 through nucleotide 30 of SEQ ID NO:16, and the last codon spans from nucleotide 1237 through nucleotide 1239. The region encoding P_(ca1)PB2-N₄₀₄, designated nei_(ca1)PB2-N₁₂₁₄, and having a coding strand comprising nucleotides 28 to 1239 of SEQ ID NO:16, is represented by SEQ ID NO:18.

PCR amplification of a second nucleic acid molecule encoding a cold-adapted equine influenza PB2-N protein in the same manner resulted in molecules nei_(ca2)PB2-N₁₂₄₁, identical to nei_(ca1)PB2-N₁₂₄₁, and nei_(ca2)PB2-N₁₂₁₄, identical to nei_(ca1)PB2-N₁₂₁₄. C. Comparison of the nucleic acid sequences of the coding strands of nei_(wt)PB2-N₁₂₄₁ (SEQ ID NO:13) and nei_(ca1)PB2-N₁₂₄₁ (SEQ ID NO:16) by DNA alignment reveals the following difference: a T to C base shift at base 370. Comparison of the amino acid sequences of proteins P_(wt)PB2-N₄₀₄ (SEQ ID NO:14) and P_(ca1)PB2-N₄₀₄ (SEQ ID NO:17) reveals the following difference: a Y to H shift at amino acid 124 relating to the a T to C shift at base 370 in the DNA sequence.

EXAMPLE 14

This example describes the cloning and sequencing of equine influenza PB2 protein (RNARNA polymerase) nucleic acid molecules corresponding to the C-terminal portion of the protein, for wild type or cold-adapted equine influenza viruses.

A. Nucleic acid molecules encoding wild type or cold-adapted equine influenza virus PB2-C proteins were produced as follows. A PCR product containing the C-terminal portion of the equine PB2 gene was produced by PCR amplification using from equine influenza virus DNA and primers w572 and w573, designated SEQ ID NO:38 and SEQ ID NO:39, respectively. A nucleic acid molecule of 1233 nucleotides encoding a wild type PB2-C protein, denoted nei_(wt)PB2-C₁₂₃₃, with a coding strand having a nucleic acidic sequence designated SEQ ID NO:19 was produced by further PCR amplification using the above-described PCR product as a template and cloned as described in Example 11B. Plasmid DNA was purified and sequenced as in Example 11A, except that different primers were used in the sequencing kits. T7 and REV were used in one instance, efPB2-a1, designated SEQ ID NO:40 and efPB2-s1, designated SEQ ID NO:41 were used in another instance, and efPB2-a2, designated SEQ ID NO:42 and efPB2-s2, designated SEQ ID NO:43 were used in another instance.

Translation of SEQ ID NO:19 indicates that nucleic acid molecule nei_(wt1)PB2-C₁₂₃₃ encodes a C-terminal portion of influenza PB2 protein of about 398 amino acids, referred to herein as P_(wt)PB2-C₃₉₈, having amino acid sequence SEQ ID NO:20, assuming an open reading frame having a first codon spans from nucleotide 3 through nucleotide 5 and a termination codon which spans from nucleotide 1197 through nucleotide 1199 of SEQ ID NO:19. Because SEQ ID NO:19 is only a partial gene sequence, it does not contain an initiation codon. The region encoding P_(wt)PB2-C₃₉₈, designated nei_(wt)PB2-C₁₁₉₄, and having a coding strand comprising nucleotides 3 to 1196 of SEQ ID NO:19 is represented by SEQ ID NO:21.

PCR amplification of a second nucleic acid molecule encoding a wild type equine influenza PB2-N protein in the same manner resulted in a nucleic acid molecule of 1232 nucleotides denoted nei_(wt2)PB2-N₁₂₃₂, with a coding strand with a sequence designated SEQ ID NO:22. nei_(wt2)PB2-N₁₂₃₂ is identical to nei_(wt1)PB2-C₁₂₃₃, expect that nei_(wt2)PB2-N₁₂₃₂ lacks one nucleotide on the 5′-end. Translation of SEQ ID NO:22 indicates that nucleic acid molecule nei_(wt1)PB2-C₁₂₃₃ also encodes P_(wt)PB2-C₃₉₈ (SEQ ID NO:20), assuming an open reading frame having a first codon which spans from nucleotide 2 through nucleotide 4 and a termination codon spans from nucleotide 1196 through nucleotide 1198 of SEQ ID NO:22. Because SEQ ID NO:22 is only a partial gene sequence, it does not contain an initiation codon. The nucleic acid molecule having a coding strand comprising nucleotides 2 to 1195 of SEQ ID NO:22, denoted nei_(wt2)PB2-C₁₁₉₄, is identical to SEQ ID NO:21.

B. A nucleic acid molecule of 1232 nucleotides encoding a C-terminal portion of influenza PB2 cold-adapted equine influenza virus protein, denoted nei_(ca1)PB2-C₁₂₃₂, and having a coding strand having a sequence designated SEQ ID NO:23 was produced as described in as in Example 14, part A, except that the pCR®-Blunt cloning vector was used.

Translation of SEQ ID NO:23 indicates that nucleic acid molecule nei_(ca1)PB2-C₁₂₃₂ encodes a C-terminal portion of equine influenza PB-2 protein of about 398 amino acids, referred to herein as P_(ca1)PB2-C₃₉₈, having amino acid sequence SEQ ID NO:24, assuming an open reading frame having a first codon which spans from nucleotide 2 through nucleotide 4 and a termination codon spans from nucleotide 1196 through nucleotide 1198 of SEQ ID NO:23. Because SEQ ID NO:23 is only a partial gene sequence, it does not contain an initiation codon. The region encoding P_(ca1)PB2-C₃₉₈, designated nei_(ca1)PB2-C1194, and having a coding strand comprising nucleotides 2 to 1195 of SEQ ID NO:23, is represented by SEQ ID NO:25. PCR amplification of a second nucleic acid molecule encoding a cold-adapted equine influenza PB2-C protein in the same manner resulted in molecules nei_(ca2)PB2-C₁₂₃₁, containing one less nucleotide at the 3′end than nei_(ca1)PB2-N₁₂₄₁; and nei_(ca2)PB2-N₁₂₁₄, identical to nei_(ca1)PB2-N₁₂₁₄.

C. Comparison of the nucleic acid sequences of the coding strands of nei_(wt1)PB2-C₁₂₃₃ (SEQ ID NO:19) and nei_(ca1)PB2-C₁₂₃₂ (SEQ ID NO:23) by DNA alignment reveals the following differences: an A to C base shift at base 153 of SEQ ID NO:19, and a G to A base shift at base 929 of SEQ ID NO:19. Comparison of the amino acid sequences of proteins P_(wt)PB2-C₃₉₈ (SEQ ID NO:20) and P_(ca1)PB2-₃₉₈ (SEQ ID NO:24) reveals thee following difference: a K to Q shift at amino acid 51 when relating to the an A to C base shift at base 153 in the DNA sequences. There is no amino acid shift resulting from the G to A base shift at base 929.

EXAMPLE 15

This example describes the cloning and sequencing of equine influenza PB2 protein (RNA-directed RNA polymerase) nucleic acid molecules for wild type or cold-adapted equine influenza viruses.

A. Nucleic acid molecules encoding wild type or cold-adapted equine influenza virus PB2 proteins were produced as follows. The wild type or cold-adapted equine influenza genes were cloned in two fragments, the N-terminal portion was produced as in Example 13 and the C-terminal portion of the gene was produced as in Example 14.

The DNA sequence for the wild type equine influenza PB2 gene was generated by combining the consensus sequences for the wild type PB2-N protein, denoted nei_(wt)PB2-N₁₂₄₁ (SEQ ID NO:13) with the gene fragments for the wild type PB2-C protein, denoted nei_(wt1)PB2-C₁₂₃₃ (SEQ ID NO:19) and nei_(wt2)PB2-C₁₂₃₂ (SEQ ID NO:22). The result of combining the consensus sequences from the N-terminal and C-terminal portions of the PB2 wild type influenza virus yielded a complete DNA sequence denoted nei_(wt)PB2₂₃₄₁ (SEQ ID NO:44). Translation of SEQ ID NO:44 indicates that the nucleic acid molecule nei_(wt)PB2₂₃₄₁ encodes a full length equine influenza PB2 protein of about 759 amino acids referred to herein as Pei_(wt)PB2₇₅₉, having amino acid sequence SEQ ID NO:45, assuming an open reading frame in which the initiation codon spans from nucleotide 28 through nucleotide 30 of SEQ ID NO:44 and the termination codon spans from nucleotide 2305 through nucleotide 2307 of SEQ ID NO:44. The region encoding Pei_(wt)PB2₇₅₉, designated nei_(wt)PB2₂₂₇₇, and having a coding strand comprising nucleotides 28 to 2304 of SEQ ID NO:44, is SEQ ID NO:46.

B. A DNA sequence of 2341 nucleotides encoding a cold-adapted equine influenza virus PB2, denoted nei_(ca1)PB2₂₃₄₁, with a sequence denoted SEQ ID NO:47 was produced by combining the sequences for the N-terminal and C-terminal portions of the PB2 cold-adapted equine influenza gene. The clones for the N-terminal sequences are denoted nei_(ca1)PB2-N₁₂₄₁ and nei_(ca2)PB2-N₁₂₄₁ which are identical and are represented by SEQ ID NO:16. The clones for the C-terminal sequences are denoted nei_(ca1)PB2-C₁₂₃₂ and nei_(ca2)PB2-C₁₂₃₁, represented by SEQ ID NO:23.

Translation of SEQ ID NO:47 indicates that nucleic acid molecule nei_(ca1)PB2₂₃₄₁ encodes a full-length equine influenza PB2 protein of about 759 amino acids, referred to herein as Pei_(ca1)PB2₇₅₉, having amino acid sequence SEQ ID NO:48, assuming an open reading frame in which the initiation codon spans from nucleotide 28 through nucleotide 30 of SEQ ID NO:47 and the termination codon spans from nucleotide 2305 through nucleotide 2307 of SEQ ID NO:47. The region encoding Pei_(ca1)PB2₇₅₉ designated nei_(ca1)PB2₂₂₇₇ and having a coding strand comprising nucleotides 28 to 2304 of SEQ ID NO 49.

C. Comparison of the nucleic acid sequences of the coding strands of nei_(wt)PB₂₃₄₁ (SEQ ID NO:44) and nei_(ca1)PB2₂₃₄₁ (SEQ ID NO:47) by DNA alignment reveals the following differences: a T to C base shift at base 370, an A to C base shift at base 1261, and a G to A base shift at base 2037. Comparison of the amino acid sequences of proteins Pei_(wt)PB2₇₅₉ (SEQ ID NO:45) and Pei_(ca1)PB2₇₅₉ (SEQ ID NO:48) reveals the following differences: a Y to H shift at amino acid 124 relating to the a T to C shift at base 370 in the DNA sequence, a K to Q shift at amino acid 421 relating to the A to C shift at base 1261 in the DNA sequence. The third nucleotide shift at base 2037 does not result in an amino acid shift at amino.

EXAMPLE 16

This example describes the cloning and sequencing of equine influenza NS (nonstructural) protein nucleic acid molecules for wild type or cold-adapted equine influenza viruses.

A. Nucleic acid molecules encoding wild type or cold-adapted equine influenza virus NS proteins were produced as follows. A PCR product containing an equine NS gene was produced by PCR amplification from equine influenza virus DNA and primers w586 and w587, designated SEQ ID NO:59 and SEQ ID NO:60₇, respectively. A nucleic acid molecule of 891 nucleotides encoding a wild-type NS protein, denoted nei_(wt)NS₈₉₁, with a coding strand having a nucleic acid sequence designated SEQ ID NO:50 was produced by further PCR amplification using the above-described PCR product as a template and cloned into pCR 2.1® TA cloning vector as described in Example 11A. Plasmid DNA was purified and sequenced as in Example 11A, except that primers used in the sequencing kits were only T7 and REV were used in the sequencing kits.

Translation of SEQ ID NO:50 indicates that nucleic acid molecule nei_(wt1)NS₈₉₁ encodes a full-length equine influenza NS protein of about 230 amino acids, referred to herein as Pei_(wt1)NS₂₃₀, having amino acid sequence SEQ ID NO:51, assuming an open reading frame in which the initiation codon spans from nucleotide 27 through nucleotide 29 of SEQ ID NO:50 and the termination codon spans from nucleotide 717 through nucleotide 719 of SEQ ID NO:50. The region encoding Pei_(wt1)NS₂₃₀, designated nei_(wt1)NS₆₉₀, and having a coding strand comprising nucleotides 27 to 716 of SEQ ID NO:50 is represented by SEQ ID NO:52.

PCR amplification of a second nucleic acid molecule encoding a wild type equine influenza NS protein in the same manner resulted in molecules nei_(wt2)NS₈₉₁, identical to nei_(wt1)NS₈₉₁ in the coding region i.e. nei_(wt2)NS₆₉₀, is identical to nei_(wt1)NS₆₉₀. nei_(wt2)NS₈₉₁ differs from nei_(wt1)NS₈₉₁ in one nucleotide at base 827 (G to A) which is 111 bases downstream from the stop codon. PCR amplification of a third nucleic acid encoding a wild type equine influenza NS protein in the same manner resulted in a nucleic acid molecule of 888 nucleotides denoted nei_(wt3)NS₈₈₈, with a coding strand with a nucleic acid sequence designated SEQ ID NO:53. nei_(wt3)NS₈₈₈ is identical to nei_(wt1)NS₈₉₁, except that nei_(wt3)NS₈₈₈, lacks two nucleotides on the 5′ end and one nucleotide on the 3′ end. Translation of SEQ ID NO:53 indicates that nucleic acid molecule nei_(wt3)NS₈₈₈ also encodes Pei_(wt1)NS₂₃₀ (SEQ ID NO:51), assuming an open reading frame having an initiation codon which spans from nucleotide 25 through nucleotide 27 of SEQ ID NO:53 and a termination codon which spans from nucleotide 715 through nucleotide 717 of SEQ ID NO:53. The nucleic acid molecule having a coding strand comprising nucleotides 25 to 714 of SEQ ID 53, denoted nei_(wt3)NS₆₉₀, is identical to SEQ ID NO:52.

PCR amplification of a fourth nucleic acid of 468 nucleotides encoding a C-terminal portion of the wild type equine influenza NS protein, denoted nei_(wt4)NS₄₆₈ and having a coding sequence designated SEQ ID NO:54 was produced. Translation of SEQ ID NO:54 indicates that nucleic acid molecule nei_(wt4)NS₄₆₈ encodes a C-terminal portion of equine influenza NS protein of about 97 amino acids, referred to herein as Pei_(wt4)NS₉₇, having amino acid sequence SEQ ID NO:55, assuming an open reading frame having a first codon which spans from nucleotide 3 to 5 of SEQ ID NO:54, and a termination codon spans from nucleotide 294 through 296 of SEQ ID NO:54. Because SEQ ID NO:54 is only a partial gene sequence, it does not contain an initiation codon. The region encoding Pei_(wt4)NS₉₇, designated nei_(wt4)NS₂₉₃, and having a coding strand comprising nucleotides 1 to 293 of SEQ ID NO:54, is represented by SEQ ID NO:56.

B. A nucleic acid molecule of 888 nucleotides encoding a cold-adapted equine influenza virus NS protein, denoted nei_(ca1)NS₈₈₈, with a coding strand having a sequence designated SEQ ID NO:57 was produced and sequenced as described in Example 16, part A.

Translation of SEQ ID NO:57 indicates that nucleic acid molecule nei_(ca1)NS₈₈₈ encodes a full-length equine influenza NS protein of about 230 amino acids, referred to herein as Pei_(ca1)NS₂₃₀, having amino acid sequence SEQ ID NO:58, assuming an open reading frame in which the initiation codon spans from nucleotide 27 through nucleotide 29 of SEQ ID NO:57 and the termination codon spans from nucleotide 717 through nucleotide 719 of SEQ ID NO:57. The region encoding Pei_(ca1)NS₂₃₀, designated nei_(ca1)NS₆₉₀, and having a coding strand comprising nucleotides 27 to 716 of SEQ ID NO:57, is represented by SEQ ID NO:59.

PCR amplification of a second nucleic acid molecule encoding a cold-adapted equine influenza NS protein in the same manner resulted in molecules nei_(ca2)NS₈₈₇, containing one less nucleotide at the 3′ end than nei_(ca1)NS₈₈₈; the coding region nei_(ca2)NS₆₉₀ is identical to nei_(ca1)NS₆₉₀.

C. Comparison of the nucleic acid sequences of the coding strands of nei_(wt)NS₈₉₁ (SEQ ID NO:50) and nei_(ca1)NS₈₈₈ (SEQ ID NO:57) by DNA alignment reveals the following difference: an A to G shift at base 827 which is 111 bases downstream from the stop codon. The 3′ fragment encoding nei_(wt4)NS₄₆₈ (SEQ ID NO:54) has one shift T to C found at base 633 relative to the full-length consensus sequence. Comparison of the amino acid sequences of proteins Pei_(wt)NS₂₃₀ (SEQ ID NO:51) and Pei_(ca1)NS₂₃₀ (′SEQ ID NO:58) reveals that there are no differences between amino acid sequences of the wild type and cold-adapted proteins.

EXAMPLE 17

This example describes the cloning and sequencing of equine influenza PB1 protein (RNA-directed RNA polymerase 1) nucleic acid molecules corresponding to the N-terminal portion of the protein, for wild type or cold-adapted equine influenza viruses.

A. Nucleic acid molecules encoding wild type or cold-adapted equine influenza virus PB1-N proteins were produced as follows. A PCR product containing an N-terminal portion of the equine PB1 gene was produced by PCR amplification from equine influenza virus DNA, and primers T7 and REV. A nucleic acid molecule of 1229 nucleotides encoding,a wild type PB1-N protein, denoted nei_(wt1)PB1-N₁₂₂₉, with a coding strand having a nucleic acid sequence designated SEQ ID NO:62 was produced by further PCR amplification using the above described PCR product as a template and cloned as described in Example 11B . Plasmid DNA was purified and sequenced as in Example 11B, except that only T7 and REV primers were used in the sequencing kits.

Translation of SEQ ID NO:62 indicates that nucleic acid molecule nei_(wt1)PB1-N₁₂₂₉ encodes an N-terminal portion of influenza PB1 protein of about 398 amino acids, referred to herein as Pei_(wt1)PB1-N₃₉₈, having amino acid sequence SEQ ID NO:63, assuming an open reading frame in which the initiation codon spans from nucleotide 36 through nucleotide 38 of SEQ ID NO:62, and the last codon spans from nucleotide 1227 through nucleotide 1229 of SEQ ID NO:62. The region encoding Pei_(wt1)PB1-N₃₉₈, designated nei_(wt1)PB1-N₁₁₉₄, and having a coding strand comprising nucleotides 36 to 1229 of SEQ ID NO:62 is represented by SEQ ID NO:64.

PCR amplification of a second nucleic acid molecule encoding a wild type equine influenza PB1-N protein in the same manner resulted in a nucleic acid molecule of 673 nucleotides denoted nei_(wt1)PB1-N₆₇₃, with a coding strand with a sequence designated SEQ ID NO:65. Translation of SEQ ID NO:65 indicates that nucleic acid molecule nei_(wt2)PB1-N₆₇₃ encodes Pei_(wt2)PB1-N₂₁₂ (SEQ ID NO:66), assuming an open reading frame having an initiation codon which spans from nucleotide 36 through nucleotide 38 of SEQ ID NO:65 and a last codon which spans from nucleotide 671 through nucleotide 673 of SEQ ID NO:65. Because SEQ ID NO:65 is only a partial gene sequence, it does not contain a stop codon. The nucleic acid molecule having a coding strand comprising nucleotides 36 to 671 of SEQ ID NO:65, denoted nei_(wt2)PB1-N₆₃₆, is designated SEQ ID NO:67.

B. A nucleic acid molecule of 1225 nucleotides encoding an N-terminal portion of influenza PB1 cold-adapted equine influenza virus PB1-N protein, denoted nei_(ca1)PB1-N₁₂₂₅, with a coding strand having a sequence designated SEQ ID NO:68 was produced, and sequenced as described in as in Example 17, part A.

Translation of SEQ ID NO:68 indicates that nucleic acid molecule nei_(ca1)PB1-N₁₂₂₅ encodes an N-terminal portion of equine influenza PB-1 protein of about 395 amino acids, referred to herein as Pei_(ca1)PB1-N₃₉₅, having amino acid sequence SEQ ID NO:69, assuming an open reading frame in which the initiation codon spans from nucleotide 34 through nucleotide 36 of SEQ ID NO:68, and a last codon which spans from nucleotide 1216 through nucleotide 1218 of SEQ ID NO:68. The region encoding Pei_(ca1)PB1-N₃₉₅, designated nei_(ca1)PB1-N₁₁₈₅, and having a coding strand comprising nucleotides 34 to 1218 of SEQ ID NO:68, is represented by SEQ ID NO:70.

PCR amplification of a second nucleic acid molecule encoding a cold-adapted equine influenza PB1-N protein in the same manner resulted in molecules nei_(ca2)PB1-N₁₂₂₁, designated SEQ ID NO:71, containing four less nucleotides at the 5′ end than nei_(ca1)PB1-N₁₂₂₅; the coding region nei_(ca2)PB1-N₁₁₈₅, is identical to nei_(ca1)PNB1-N₁₁₈₅.

C. Comparison of the nucleic acid sequences of the coding strands of nei_(wt)PB1-N₁₂₂₉ (SEQ ID NO:62) and nei_(ca1)PB1-N₁₂₂₅ (SEQ ID NO:68) by DNA alignment reveals no differences in the coding regions. Comparison of the amino acid sequences of proteins Pei_(wt)PB1-N₃₉₅ (SEQ ID NO:63) and Pei_(ca1)PB1-N₃₉₅ (SEQ ID NO:69) also reveals no differences.

EXAMPLE 18

This example describes the cloning and sequencing of equine influenza PB1 protein (RNA-directed RNA polymerasel) nucleic acid molecules corresponding to the C-terminal portion of the protein, for wild type or cold-adapted equine influenza viruses.

A. Nucleic acid molecules encoding wild type or cold-adapted equine influenza virus PB1-C proteins were produced as follows. A PCR product containing an C-terminal portion of the equine PB1 gene was produced by PCR amplification from equine influenza virus DNA, and primer w569 designated SEQ ID NO:102. A nucleic acid molecule of 1234 nucleotides encoding a wild type PB1-C protein, denoted nei_(wt1)PB1-C₁₂₃₄, with a coding strand having a nucleic acid sequence designated SEQ ID NO:85 was produced by further PCR amplification using the above described PCR product as a template and cloned as described in Example 11B. Plasmid DNA was purified and sequenced as in Example 11A, except that different primers were used in the sequencing kits. T7, REV, w569, efPB1-a1, designated SEQ ID NO:97, efPB1-a2, designated SEQ ID NO:98, efPB1-s1, designated SEQ ID NO:99, efPB1-s2, designated SEQ ID NO:100, and efPB1-s3, designated SEQ ID NO:101 were used in one instance, T7, REV, efPB1-a1, efPB1-a2, efPB1-s1, efPB1-s2, and efPB1-s3 were used in another instance and T7 and REV were used in another instance.

Translation of SEQ ID NO:85 indicates that nucleic acid molecule nei_(wt1)PB1-C₁₂₃₄ encodes an C-terminal portion of influenza PB1 protein of about 396 amino acids, referred to herein as Pei_(wt1)PB1-C₃₉₆, having amino acid sequence SEQ ID NO:86, assuming an open reading frame in which the first codon spans from nucleotide 1 through, nucleotide 3 of SEQ ID NO:85 and a termination codon which spans from nucleotide 1189 through nucleotide 1191 of SEQ ID NO:85. Because SEQ ID NO:85 is only a partial gene sequence, it does not contain an initiation codon. The region encoding Pei_(wt1)PB1-C₃₉₆, designated nei_(wt1)PB1-C₁₁₈₈, and having a coding strand comprising nucleotides 1 to 1188 of SEQ ID NO:85 is represented by SEQ ID NO:87.

PCR amplification of a second nucleic acid molecule encoding a wild type equine influenza PB1-C protein in the same manner resulted in a nucleic acid molecule of 1240 nucleotides denoted nei_(wt2)PB1-C₁₂₄₀ with a coding strand with a sequence designated SEQ ID NO:88. Translation of SEQ ID NO:88 indicates that nucleic acid molecule nei_(wt2)PB1-N₁₂₄₀ encodes a molecule designated Pei_(wt2)PB1-C₃₉₆ (SEQ ID NO:89) which differs from Pei_(wt1)PB1-C₃₉₆ (SEQ ID NO:85) in one nucleotide. Nucleotide 382 of nei_(wt1)PB1-C₁₂₃₄, i.e. nucleotide 382 of nei_(wt1)PB1-C₁₁₈₈ was A, while nucleotide 389 of nei_(wt2) PB1-C₁₂₄₀, i.e. nucleotide 382 of nei_(wt)2PB1-C₁₁₈₈ was T. Translation of nei_(wt2)PB1-C₁₂₄₀ results in an amino acid change of T to S.

B. A nucleic acid molecule of 1241 nucleotides encoding an C-terminal portion of influenza PB1 cold-adapted equine influenza virus PB1-C protein, denoted nei_(ca1)PB1-C₁₂₄₁, with a coding strand having a sequence designated SEQ ID NO:91 was produced, and sequenced as described in as in Example 18, part A.

Translation of SEQ ID NO:91 indicates that nucleic acid molecule nei_(ca1)PB1-C₁₂₄₁ encodes an C-terminal portion of equine influenza PB-1 protein of about 396 amino acids, referred to herein as Pei_(ca1)PB1-C₃₉₆, having amino acid sequence SEQ ID NO:92, assuming an open reading frame in which the first codon spans from nucleotide 8 through nucleotide 10 of SEQ ID NO:91 and a termination codon that spans from nucleotide 1196 through nucleotide 1198 of SEQ ID NO:91. Because SEQ ID NO:91 is only a partial gene sequence, it does not contain an initiation codon. The region encoding Pei_(ca)1PB1-C₃₉₆, designated nei_(ca1)PB1-C₁₁₈₈, and having a coding strand comprising nucleotides 8 to 1195 of SEQ ID NO:91, is represented by SEQ ID NO:93.

PCR amplification of a second nucleic acid molecule encoding a cold-adapted equine influenza PB1-C protein in the same manner resulted in a nucleic acid molecule of 1241 nucleotides denoted nei_(ca2)PB1-C₁₂₄₁, with a coding strand with a sequence designated SEQ ID NO:94. Translation of SEQ ID NO:94 indicates that nucleic acid molecule nei_(ca2)PB1-C₁₂₄₁ encodes a molecule designated Pei_(ca2)PB1-C₃₉₆ (SEQ ID NO:95) which differs from Pei_(ca1)PB1-C₃₉₆ (SEQ ID NO:92) in one nucleotide. Nucleotide 1044 of nei_(ca1)PB1-C₁₂₄₁, i.e. nucleotide 1037 of nei_(ca1)PB1-N₁₁₈₈ was A, while nucleotide 1044 of nei_(ca2)PB1-C₁₂₄₁, i.e. nucleotide 1037 of nei_(ca2)PB1-C₁₁₈₈ was G. Translation of nei_(ca2)PB1-C₁₂₄₁ results in an amino acid change of R to K.

C. Comparison of the nucleic acid sequences of the coding strands of nei_(wt1)PB1-C₁₂₃₄ (SEQ ID NO:85) and nei_(ca1)PB1-C₁₂₄₁ (SEQ ID NO:91) by DNA alignment reveals the following differences: a C to T shift at base 600 of SEQ ID NO:85, and a T to A shift at base 603 of SEQ ID NO:85. Comparison of the amino acid sequences of proteins Pei_(wt1)PB1-C₃₉₆ (SEQ ID NO:86) and Pei_(ca1)PB1-N₃₉₆ (SEQ ID NO:92) reveals the following difference: a H to Q amino acid shift 203 when relating to the T to A, base shift at base 603 in the DNA sequences. There is no amino acid shift resulting from the C to T base shift at base 600.

EXAMPLE 19

This example describes the cloning and sequencing of equine influenza PB1 protein (RNA-directed RNA polymerase) nucleic acid molecules for wild type or cold-adapted equine influenza viruses.

A. Nucleic acid molecules encoding wild type or cold-adapted equine influenza virus PB1 proteins were produced as follows. The wild type or cold-adapted equine influenza genes were cloned in two fragments, the N-terminal portion was produced as in Example 17 and the C-terminal portion of the gene was produced as in Example 18.

The DNA sequence for the wild type equine influenza PB1 gene was generated by combining the sequences for the wild type PB1-N protein, nei_(wt1)PB1-N₁₂₂₉ (SEQ ID NO:62) and nei_(wt2)PB1-N₆₇₃ (SEQ ID NO:65) with the gene fragments for the wild type PB1-C protein, denoted nei_(wt1)PB1-C₁₂₃₄ (SEQ ID NO:85) and nei_(wt2)PB1-C₁₂₄₀ (SEQ ID NO:88). The result of combining the N-terminal and C-terminal portions of the PB1 wild type influenza virus yielded a complete DNA sequence of 2341 nucleotides denoted nei_(wt)PB1₂₃₄₁ (SEQ ID NO:103). Translation of SEQ ID NO:103 indicates that the nucleic acid molecule nei_(wt)PB2₂₃₄₁ encodes a full length equine influenza PB1 protein of about 757 amino acids referred to herein as Pei_(wt)PB1₇₅₇, having amino acid sequence SEQ ID NO:104, assuming an open reading frame in which the initiation codon spans from nucleotide 25 through nucleotide 27 of SEQ ID NO:103 and tie termination codon spans from nucleotide 2293 through nucleotide 2295 of SEQ ID NO:103. The region encoding Pei_(wt)PB1₇₅₇ designated nei_(wt)PB1₂₂₇₁, and having a coding strand comprising nucleotides 25 to 2292 of SEQ ID NO:103, is SEQ ID NO:105.

B. A DNA sequence of 2341 nucleotides encoding a cold-adapted equine influenza virus PB1, denoted nei_(ca1)PB1₂₃₄₁, with a sequence denoted SEQ ID NO:106 was produced by combining the sequences for the N-terminal and C-terminal portions of the PB1 cold-adapted equine influenza gene. The clones for the N-terminal sequences are denoted nei_(ca1)PB1-N₁₂₂₅(SEQ ID NO:68) and nei_(ca2)PB1-N₁₂₂₁ (SEQ ID NO: 71). The clones for the C-terminal sequences are denoted nei_(ca1)PB1-C₁₂₄₁ (SEQ ID NO:91) and nei_(ca1)PB1-C₁₂₄₁, (SEQ ID NO:94).

Translation of SEQ ID NO:106 indicates that nucleic acid molecule nei_(ca1)PB1₂₃₄₁ encodes a full-length equine influenza PB1 protein of about 757 amino acids, referred to herein as Pei_(ca1)PB1₇₅₇, having amino acid sequence SEQ ID NO:107, assuming an open reading frame in which the initiation codon spans from nucleotide 25 through nucleotide 27 SEQ ID NO:106 and the termination codon spans from nucleotide 2296 through nucleotide 2298 of SEQ ID NO:106. The region encoding Pei_(ca1)PB1₇₅₇ designated nei_(ca1)PB1₂₂₇₁ and having a coding strand comprising nucleotides 25 to 2295 of SEQ ID NO:108.

C. Comparison of the nucleic acid sequences of the coding strands of nei_(wt)PB1₂₃₄₁ (SEQ ID NO:103) and nei_(ca1)PB1₂₃₄₁ (SEQ ID NO:106) by DNA alignment reveals the following differences: a C to T base shift at base 1683, and a T to A base shift at base 1686. Comparison of the amino acid sequences of proteins Pei_(wt)PB1₇₅₇ (SEQ ID NO:104) and Pei_(ca1)PB1₇₅₇ (SEQ ID NO:107) reveals the following differences: no shift in base C at amino acid 561 relating to the C to T shift at base 1683, and a H to Q shift at amino acid 562 relating to the a T to A shift at base 1683 in the DNA sequence.

EXAMPLE 20

This example describes the cloning and sequencing of equine influenza PA protein (RNA polymerase A) nucleic acid molecules corresponding to the C-terminal portion of the protein, for wild type or cold-adapted equine influenza viruses.

A. Nucleic acid molecules encoding wild type or cold-adapted equine influenza virus PA-C proteins were produced as follows. A PCR product containing the C-terminal portion of the equine PA gene was produced by PCR amplification using from equine influenza virus DNA and primers C+PA and C−PA, designated SEQ ID NO:83 and SEQ ID NO:84 respectively. A nucleic acid molecule of 1228 nucleotides encoding a wild type PA-C protein, denoted nei_(wt1)PA-C₁₂₂₈, with a coding strand having a nucleic acid sequence designated SEQ ID NO:76 was produced by further PCR amplification using the above-described PCR product as a template and cloned as described in Example 11B . Plasmid DNA was purified and sequenced as in Example 11A, except that different primers were used in the sequencing kits. T7 and REV were used in one instance; PAC-1, designated SEQ ID NO:72, PAC-2, designated SEQ ID NO:73, PAC-3, designated SEQ ID NO:74, PAC-4, designated SEQ ID NO:75, T7 and REV were used in another instance; and PAC-1, PAC-2, T7 and REV were used in another instance.

Translation of SEQ ID NO:76 indicates that nucleic acid molecule nei_(wt1)PA-C₁₂₂₈ encodes a C-terminal portion of influenza PA protein of about 388 amino acids, referred to herein as Pei_(wt1)PA-C₃₈₈, having amino acid sequence SEQ ID NO:77, assuming an open reading frame having a first codon spans from nucleotide 3 through nucleotide 5 of SEQ ID NO:76 and a termination codon which spans from nucleotide 1167 through nucleotide 1169 of SEQ ID NO:76. Because SEQ ID NO:76 is only a partial gene sequence, it does not contain an initiation codon. The region encoding Pei_(wt1)PA-C₃₈₈, designated nei_(wt1)PA-C₁₁₆₄, and having a coding strand comprising nucleotides 3 to 1166 of SEQ ID NO:76 is represented by SEQ ID NO:78.

PCR amplification of a second nucleic acid molecule encoding a wild type equine influenza PA-C protein in the same manner resulted in a nucleic acid molecule of 1223 nucleotides denoted nei_(wt2)PA-C₁₂₂₃, with a coding strand with a sequence designated SEQ ID NO:79. nei_(wt2)PA-C₁₂₂₃ is identical to nei_(wt1)PA-C₁₂₂₈, with the exception of a T to C base shift at base 753 and that nei_(wt2)PA-C₁₂₂₃ lacks five nucleotides on the 3′-end. Translation of SEQ ID NO:79 indicates that nucleic acid molecule nei_(wt2)PA-C₁₂₂₃ also encodes Pei_(wt1)PA-C₃₈₈ (SEQ ID NO:77), assuming an open reading frame having a first codon which spans from nucleotide 3 through nucleotide 5 of SEQ ID NO:79 and a termination codon which spans from nucleotide 1167 through nucleotide 1169 of SEQ ID NO:79. Because SEQ ID NO:79 is only a partial gene sequence, it does not contain an initiation codon. The nucleic acid molecule having a coding strand comprising nucleotides 3 to 1166 of SEQ ID NO:79, denoted nei_(wt2)PA-C₁₂₂₃, is identical to SEQ ID NO 78.

B. A nucleic acid molecule of 1233 nucleotides encoding a C-terminal portion of influenza PA-C cold-adapted equine influenza virus protein, denoted nei_(ca1)PA-C₁₂₃₃, and having al coding strand having a sequence designated SEQ ID NO:80 was produced as described in as in Example 20, part A, except that the pCR®-Blunt cloning vector was used.

Translation of SEQ ID NO:80 indicates that nucleic acid molecule nei_(ca1)PA-C₁₂₃₃ encodes a C-terminal portion of equine influenza PA protein of about 390 amino acids, referred to herein as Pei_(ca1)PA-C₃₉₀, having amino acid sequence SEQ ID NO:81, assuming an open reading frame having a first codon which spans from nucleotide 3 through nucleotide 5 of SEQ ID NO:80 and a termination codon which spans from nucleotide 1173 through nucleotide 1175 of SEQ ID NO:80. Because SEQ ID NO:80 is only a partial gene sequence, it does not contain an initiation codon. The region encoding Pei_(ca1)PA-C₃₉₀, designated nei_(ca1)PA-C₁₁₇₀, and having a coding strand comprising nucleotides 3 to 1172 of SEQ ID NO:80, is represented by SEQ ID NO:82.

PCR amplification of a second nucleic acid molecule encoding a cold-adapted equine influenza PA-C protein in the same manner resulted in molecule nei_(ca2)PA-C₁₂₃₃, containing one A to C base shift at base 953 as compared to nei_(ca1)PA-C₁₂₃₃; this base shift at does not result in an amino acid change so Pei_(ca2)PA-C₃₉₀, is identical to Pei_(ca1)PA-C₃₉₀ (SEQ ID NO:81.) C. Comparison of the nucleic acid sequences of the coding strands of nei_(wt1)PA-C₁₂₂₈ (SEQ ID NO:76) and nei_(ca1)PA-C₁₂₃₃ (SEQ ID NO:80) by DNA alignment reveals the following difference: an C to T base shift at base 753 of SEQ ID NO:80. Comparison of the amino acid sequences of proteins Pei_(wt1)PA-C₃₈₈ (SEQ ID NO:77) and Pei_(ca1)PA-₃₉₀ (SEQ ID NO:81) reveals the following difference: a W to R shift at amino acid 251 when relating to the C to T base shift at base 753 in the DNA sequences.

While various embodiments of the present invention have been described in detail, it is apparent that modifications and adaptations of those embodiments will occur to those skilled in the art. It is to be expressly understood, however, that such modifications and adaptations are within the scope of the present invention, as set forth in the following claims.

108 1 1023 DNA Equine influenza virus H3N8 CDS (25)..(780) 1 gcaaaagcag gtagatattt aaag atg agt ctt ctg acc gag gtc gaa acg 51 Met Ser Leu Leu Thr Glu Val Glu Thr 1 5 tac gtt ctc tct atc gta cca tca ggc ccc ctc aaa gcc gag atc gcg 99 Tyr Val Leu Ser Ile Val Pro Ser Gly Pro Leu Lys Ala Glu Ile Ala 10 15 20 25 cag aga ctt gaa gat gtc ttt gca ggg aag aac acc gat ctt gag gca 147 Gln Arg Leu Glu Asp Val Phe Ala Gly Lys Asn Thr Asp Leu Glu Ala 30 35 40 ctc atg gaa tgg cta aag aca aga cca atc ctg tca cct ctg act aaa 195 Leu Met Glu Trp Leu Lys Thr Arg Pro Ile Leu Ser Pro Leu Thr Lys 45 50 55 ggg att tta gga ttc gta ttc acg ctc acc gtg ccc agt gag cga gga 243 Gly Ile Leu Gly Phe Val Phe Thr Leu Thr Val Pro Ser Glu Arg Gly 60 65 70 ctg cag cgt aga cgc ttt gtc caa aat gcc ctt agt gga aac gga gat 291 Leu Gln Arg Arg Arg Phe Val Gln Asn Ala Leu Ser Gly Asn Gly Asp 75 80 85 cca aac aac atg gac aga gca gta aaa ctg tac agg aag ctt aaa aga 339 Pro Asn Asn Met Asp Arg Ala Val Lys Leu Tyr Arg Lys Leu Lys Arg 90 95 100 105 gaa ata aca ttc cat ggg gca aaa gag gtg gca ctc agc tat tcc act 387 Glu Ile Thr Phe His Gly Ala Lys Glu Val Ala Leu Ser Tyr Ser Thr 110 115 120 ggt gca cta gcc agc tgc atg gga ctc ata tac aac aga atg gga act 435 Gly Ala Leu Ala Ser Cys Met Gly Leu Ile Tyr Asn Arg Met Gly Thr 125 130 135 gtg aca acc gaa gtg gca ttt ggc ctg gta tgc gcc aca tgt gaa cag 483 Val Thr Thr Glu Val Ala Phe Gly Leu Val Cys Ala Thr Cys Glu Gln 140 145 150 atc gct gat tcc cag cat cga tct cac agg cag atg gtg aca aca acc 531 Ile Ala Asp Ser Gln His Arg Ser His Arg Gln Met Val Thr Thr Thr 155 160 165 aac cca tta atc aga cat gaa aac aga atg gta tta gcc agt acc acg 579 Asn Pro Leu Ile Arg His Glu Asn Arg Met Val Leu Ala Ser Thr Thr 170 175 180 185 gct aaa gcc atg gag cag atg gca ggg tcg agt gag cag gca gca gag 627 Ala Lys Ala Met Glu Gln Met Ala Gly Ser Ser Glu Gln Ala Ala Glu 190 195 200 gcc atg gag gtt gct agt aag gct agg cag atg gtr cag gca atg aga 675 Ala Met Glu Val Ala Ser Lys Ala Arg Gln Met Xaa Gln Ala Met Arg 205 210 215 acc att ggg acc cac cct agc tcc agt gcc ggt ttg aaa gat gat ctc 723 Thr Ile Gly Thr His Pro Ser Ser Ser Ala Gly Leu Lys Asp Asp Leu 220 225 230 ctt gaa aat ttg cag gcc tac cag aaa cgg atg gga gtg caa atg cag 771 Leu Glu Asn Leu Gln Ala Tyr Gln Lys Arg Met Gly Val Gln Met Gln 235 240 245 cga ttc aag tgatcctctc gttattgcag caagtatcat tgggatcttg 820 Arg Phe Lys 250 cacttgatat tgtggattct tgatcgcctt ttcttcaaat tcatttatcg tcgccttaaa 880 tacgggttga aaagagggcc ttctacggaa ggagtacctg agtctatgag ggaagaatat 940 cggcaggaac agcagaatgc tgtggatgtt gacgatggtc attttgtcaa catagagctg 1000 gagtaaaaaa ctaccttgtt tct 1023 2 252 PRT Equine influenza virus H3N8 misc_feature (213)..(213) The ′Xaa′ at location 213 stands for Val. 2 Met Ser Leu Leu Thr Glu Val Glu Thr Tyr Val Leu Ser Ile Val Pro 1 5 10 15 Ser Gly Pro Leu Lys Ala Glu Ile Ala Gln Arg Leu Glu Asp Val Phe 20 25 30 Ala Gly Lys Asn Thr Asp Leu Glu Ala Leu Met Glu Trp Leu Lys Thr 35 40 45 Arg Pro Ile Leu Ser Pro Leu Thr Lys Gly Ile Leu Gly Phe Val Phe 50 55 60 Thr Leu Thr Val Pro Ser Glu Arg Gly Leu Gln Arg Arg Arg Phe Val 65 70 75 80 Gln Asn Ala Leu Ser Gly Asn Gly Asp Pro Asn Asn Met Asp Arg Ala 85 90 95 Val Lys Leu Tyr Arg Lys Leu Lys Arg Glu Ile Thr Phe His Gly Ala 100 105 110 Lys Glu Val Ala Leu Ser Tyr Ser Thr Gly Ala Leu Ala Ser Cys Met 115 120 125 Gly Leu Ile Tyr Asn Arg Met Gly Thr Val Thr Thr Glu Val Ala Phe 130 135 140 Gly Leu Val Cys Ala Thr Cys Glu Gln Ile Ala Asp Ser Gln His Arg 145 150 155 160 Ser His Arg Gln Met Val Thr Thr Thr Asn Pro Leu Ile Arg His Glu 165 170 175 Asn Arg Met Val Leu Ala Ser Thr Thr Ala Lys Ala Met Glu Gln Met 180 185 190 Ala Gly Ser Ser Glu Gln Ala Ala Glu Ala Met Glu Val Ala Ser Lys 195 200 205 Ala Arg Gln Met Xaa Gln Ala Met Arg Thr Ile Gly Thr His Pro Ser 210 215 220 Ser Ser Ala Gly Leu Lys Asp Asp Leu Leu Glu Asn Leu Gln Ala Tyr 225 230 235 240 Gln Lys Arg Met Gly Val Gln Met Gln Arg Phe Lys 245 250 3 756 DNA Equine influenza virus H3N8 3 atgagtcttc tgaccgaggt cgaaacgtac gttctctcta tcgtaccatc aggccccctc 60 aaagccgaga tcgcgcagag acttgaagat gtctttgcag ggaagaacac cgatcttgag 120 gcactcatgg aatggctaaa gacaagacca atcctgtcac ctctgactaa agggatttta 180 ggattcgtat tcacgctcac cgtgcccagt gagcgaggac tgcagcgtag acgctttgtc 240 caaaatgccc ttagtggaaa cggagatcca aacaacatgg acagagcagt aaaactgtac 300 aggaagctta aaagagaaat aacattccat ggggcaaaag aggtggcact cagctattcc 360 actggtgcac tagccagctg catgggactc atatacaaca gaatgggaac tgtgacaacc 420 gaagtggcat ttggcctggt atgcgccaca tgtgaacaga tcgctgattc ccagcatcga 480 tctcacaggc agatggtgac aacaaccaac ccattaatca gacatgaaaa cagaatggta 540 ttagccagta ccacggctaa agccatggag cagatggcag ggtcgagtga gcaggcagca 600 gaggccatgg aggttgctag taaggctagg cagatggtrc aggcaatgag aaccattggg 660 acccacccta gctccagtgc cggtttgaaa gatgatctcc ttgaaaattt gcaggcctac 720 cagaaacgga tgggagtgca aatgcagcga ttcaag 756 4 1023 DNA Equine influenza virus H3N8 CDS (25)..(780) 4 gcaaaagcag gtagatattt aaag atg agt ctt ctg acc gag gtc gaa acg 51 Met Ser Leu Leu Thr Glu Val Glu Thr 1 5 tac gtt ctc tct atc tta cca tca ggc ccc ctc aaa gcc gag atc gcg 99 Tyr Val Leu Ser Ile Leu Pro Ser Gly Pro Leu Lys Ala Glu Ile Ala 10 15 20 25 cag aga ctt gaa gat gtc ttt gca ggg aag aac acc gat ctt gag gca 147 Gln Arg Leu Glu Asp Val Phe Ala Gly Lys Asn Thr Asp Leu Glu Ala 30 35 40 ctc atg gaa tgg cta aag aca aga cca atc ctg tca cct ctg act aaa 195 Leu Met Glu Trp Leu Lys Thr Arg Pro Ile Leu Ser Pro Leu Thr Lys 45 50 55 ggg att tta gga ttc gta ttc acg ctc acc gtg ccc agt gag cga gga 243 Gly Ile Leu Gly Phe Val Phe Thr Leu Thr Val Pro Ser Glu Arg Gly 60 65 70 ctg cag cgt aga cgc ttt gtc caa aat gcc ctt agt gga aac gga gat 291 Leu Gln Arg Arg Arg Phe Val Gln Asn Ala Leu Ser Gly Asn Gly Asp 75 80 85 cca aac aac atg gac aga gca gta aaa ctg tac agg aag ctt aaa aga 339 Pro Asn Asn Met Asp Arg Ala Val Lys Leu Tyr Arg Lys Leu Lys Arg 90 95 100 105 gaa ata aca ttc cat ggg gca aaa gag gtg gca ctc agc tat tcc act 387 Glu Ile Thr Phe His Gly Ala Lys Glu Val Ala Leu Ser Tyr Ser Thr 110 115 120 ggt gca cta gcc agc tgc atg gga ctc ata tac aac aga atg gga act 435 Gly Ala Leu Ala Ser Cys Met Gly Leu Ile Tyr Asn Arg Met Gly Thr 125 130 135 gtg aca acc gaa gtg gca ttt ggc ctg gta tgc gcc aca tgt gaa cag 483 Val Thr Thr Glu Val Ala Phe Gly Leu Val Cys Ala Thr Cys Glu Gln 140 145 150 atc gct gat tcc cag cat cga tct cac agg cag atg gtg aca ata acc 531 Ile Ala Asp Ser Gln His Arg Ser His Arg Gln Met Val Thr Ile Thr 155 160 165 aac cca tta atc aga cat gaa aac aga atg gta tta gcc agt acc acg 579 Asn Pro Leu Ile Arg His Glu Asn Arg Met Val Leu Ala Ser Thr Thr 170 175 180 185 gct aaa gcc atg gag cag atg gca ggg tcg agt gag cag gca gca gag 627 Ala Lys Ala Met Glu Gln Met Ala Gly Ser Ser Glu Gln Ala Ala Glu 190 195 200 gcc atg gag gtt gct agt aag gct agg cag atg gta cag gca atg aga 675 Ala Met Glu Val Ala Ser Lys Ala Arg Gln Met Val Gln Ala Met Arg 205 210 215 acc att ggg acc cac cct agc tcc agt gcc ggt ttg aaa gat gat ctc 723 Thr Ile Gly Thr His Pro Ser Ser Ser Ala Gly Leu Lys Asp Asp Leu 220 225 230 ctt gaa aat ttg cag gcc tac cag aaa cgg atg gga gtg caa atg cag 771 Leu Glu Asn Leu Gln Ala Tyr Gln Lys Arg Met Gly Val Gln Met Gln 235 240 245 cga ttc aag tgatcctctc gttattgcag caagtatcat tgggatcttg 820 Arg Phe Lys 250 cacttgatat tgtggattct tgatcgcctt ttcttcaaat tcatttatcg tcgccttaaa 880 tacggcttga aaagagggcc ttctacggaa ggagtacctg agtctatgag ggaagaatat 940 cggcaggaac agcagaatgc tgtggatgtt gacgatggtc attttgtcaa catagagctg 1000 gagtaaaaaa ctaccttgtt tct 1023 5 252 PRT Equine influenza virus H3N8 5 Met Ser Leu Leu Thr Glu Val Glu Thr Tyr Val Leu Ser Ile Leu Pro 1 5 10 15 Ser Gly Pro Leu Lys Ala Glu Ile Ala Gln Arg Leu Glu Asp Val Phe 20 25 30 Ala Gly Lys Asn Thr Asp Leu Glu Ala Leu Met Glu Trp Leu Lys Thr 35 40 45 Arg Pro Ile Leu Ser Pro Leu Thr Lys Gly Ile Leu Gly Phe Val Phe 50 55 60 Thr Leu Thr Val Pro Ser Glu Arg Gly Leu Gln Arg Arg Arg Phe Val 65 70 75 80 Gln Asn Ala Leu Ser Gly Asn Gly Asp Pro Asn Asn Met Asp Arg Ala 85 90 95 Val Lys Leu Tyr Arg Lys Leu Lys Arg Glu Ile Thr Phe His Gly Ala 100 105 110 Lys Glu Val Ala Leu Ser Tyr Ser Thr Gly Ala Leu Ala Ser Cys Met 115 120 125 Gly Leu Ile Tyr Asn Arg Met Gly Thr Val Thr Thr Glu Val Ala Phe 130 135 140 Gly Leu Val Cys Ala Thr Cys Glu Gln Ile Ala Asp Ser Gln His Arg 145 150 155 160 Ser His Arg Gln Met Val Thr Ile Thr Asn Pro Leu Ile Arg His Glu 165 170 175 Asn Arg Met Val Leu Ala Ser Thr Thr Ala Lys Ala Met Glu Gln Met 180 185 190 Ala Gly Ser Ser Glu Gln Ala Ala Glu Ala Met Glu Val Ala Ser Lys 195 200 205 Ala Arg Gln Met Val Gln Ala Met Arg Thr Ile Gly Thr His Pro Ser 210 215 220 Ser Ser Ala Gly Leu Lys Asp Asp Leu Leu Glu Asn Leu Gln Ala Tyr 225 230 235 240 Gln Lys Arg Met Gly Val Gln Met Gln Arg Phe Lys 245 250 6 756 DNA Equine influenza virus H3N8 6 atgagtcttc tgaccgaggt cgaaacgtac gttctctcta tcttaccatc aggccccctc 60 aaagccgaga tcgcgcagag acttgaagat gtctttgcag ggaagaacac cgatcttgag 120 gcactcatgg aatggctaaa gacaagacca atcctgtcac ctctgactaa agggatttta 180 ggattcgtat tcacgctcac cgtgcccagt gagcgaggac tgcagcgtag acgctttgtc 240 caaaatgccc ttagtggaaa cggagatcca aacaacatgg acagagcagt aaaactgtac 300 aggaagctta aaagagaaat aacattccat ggggcaaaag aggtggcact cagctattcc 360 actggtgcac tagccagctg catgggactc atatacaaca gaatgggaac tgtgacaacc 420 gaagtggcat ttggcctggt atgcgccaca tgtgaacaga tcgctgattc ccagcatcga 480 tctcacaggc agatggtgac aataaccaac ccattaatca gacatgaaaa cagaatggta 540 ttagccagta ccacggctaa agccatggag cagatggcag ggtcgagtga gcaggcagca 600 gaggccatgg aggttgctag taaggctagg cagatggtac aggcaatgag aaccattggg 660 acccacccta gctccagtgc cggtttgaaa gatgatctcc ttgaaaattt gcaggcctac 720 cagaaacgga tgggagtgca aatgcagcga ttcaag 756 7 1762 DNA Equine influenza virus H3N8 CDS (30)..(1724) 7 agcaaaagca ggggatattt ctgtcaatc atg aag aca acc att att ttg ata 53 Met Lys Thr Thr Ile Ile Leu Ile 1 5 cca ctg acc cat tgg gtc tac agt caa aac cca acc agt ggc aac aac 101 Pro Leu Thr His Trp Val Tyr Ser Gln Asn Pro Thr Ser Gly Asn Asn 10 15 20 aca gcc aca tta tgt ctg gga cac cat gca gta gca aat gga aca ttg 149 Thr Ala Thr Leu Cys Leu Gly His His Ala Val Ala Asn Gly Thr Leu 25 30 35 40 gta aaa aca ata act gat gac caa att gag gtg aca aat gct act gaa 197 Val Lys Thr Ile Thr Asp Asp Gln Ile Glu Val Thr Asn Ala Thr Glu 45 50 55 tta gtt cag agc att tca ata ggg aaa ata tgc aac aac tca tat aga 245 Leu Val Gln Ser Ile Ser Ile Gly Lys Ile Cys Asn Asn Ser Tyr Arg 60 65 70 gtt cta gat gga aga aat tgc aca tta ata gat gca atg cta gga gac 293 Val Leu Asp Gly Arg Asn Cys Thr Leu Ile Asp Ala Met Leu Gly Asp 75 80 85 ccc cac tgt gat gtc ttt cag tat gag aat tgg gac ctc ttc ata gaa 341 Pro His Cys Asp Val Phe Gln Tyr Glu Asn Trp Asp Leu Phe Ile Glu 90 95 100 aga agc agc gct ttc agc agt tgc tac cca tat gac atc cct gac tat 389 Arg Ser Ser Ala Phe Ser Ser Cys Tyr Pro Tyr Asp Ile Pro Asp Tyr 105 110 115 120 gca tcg ctc cgg tcc att gta gca tcc tca gga aca ttg gaa ttc aca 437 Ala Ser Leu Arg Ser Ile Val Ala Ser Ser Gly Thr Leu Glu Phe Thr 125 130 135 gca gag gga ttc aca tgg aca ggt gtc act caa aac gga aga agt gga 485 Ala Glu Gly Phe Thr Trp Thr Gly Val Thr Gln Asn Gly Arg Ser Gly 140 145 150 tcc tgc aaa agg gga tca gcc gat agt ttc ttt agc cga ctg aat tgg 533 Ser Cys Lys Arg Gly Ser Ala Asp Ser Phe Phe Ser Arg Leu Asn Trp 155 160 165 cta aca gaa tct gga aac tct tac ccc aca ttg aat gtg aca atg cct 581 Leu Thr Glu Ser Gly Asn Ser Tyr Pro Thr Leu Asn Val Thr Met Pro 170 175 180 aac aat aaa aat ttc gac aaa cta tac atc tgg ggg att cat cac ccg 629 Asn Asn Lys Asn Phe Asp Lys Leu Tyr Ile Trp Gly Ile His His Pro 185 190 195 200 agc tca aac aaa gag cag aca aaa ttg tac atc caa gaa tcg gga cga 677 Ser Ser Asn Lys Glu Gln Thr Lys Leu Tyr Ile Gln Glu Ser Gly Arg 205 210 215 gta aca gtc tca aca aaa aga agt caa caa aca ata atc cct aac atc 725 Val Thr Val Ser Thr Lys Arg Ser Gln Gln Thr Ile Ile Pro Asn Ile 220 225 230 gga tct aga ccg cgg gtc agg ggt caa tca ggc agg ata agc ata tac 773 Gly Ser Arg Pro Arg Val Arg Gly Gln Ser Gly Arg Ile Ser Ile Tyr 235 240 245 tgg acc att gta aaa cct gga gat atc cta atg ata aac agt aat ggc 821 Trp Thr Ile Val Lys Pro Gly Asp Ile Leu Met Ile Asn Ser Asn Gly 250 255 260 aac tta gtt gca ccg cgg gga tat ttt aaa ttg aaa aca ggg aaa agc 869 Asn Leu Val Ala Pro Arg Gly Tyr Phe Lys Leu Lys Thr Gly Lys Ser 265 270 275 280 tct gta atg aga tca gat gca ccc ata gac att tgt gtg tct gaa tgt 917 Ser Val Met Arg Ser Asp Ala Pro Ile Asp Ile Cys Val Ser Glu Cys 285 290 295 att aca cca aat gga agc atc ccc aac gac aaa cca ttt caa aat gtg 965 Ile Thr Pro Asn Gly Ser Ile Pro Asn Asp Lys Pro Phe Gln Asn Val 300 305 310 aac aaa gtt aca tat gga aaa tgc ccc aag tat atc agg caa aac act 1013 Asn Lys Val Thr Tyr Gly Lys Cys Pro Lys Tyr Ile Arg Gln Asn Thr 315 320 325 tta aag ctg gcc act ggg atg agg aat gta cca gaa aag caa atc aga 1061 Leu Lys Leu Ala Thr Gly Met Arg Asn Val Pro Glu Lys Gln Ile Arg 330 335 340 gga atc ttt gga gca ata gcg gga ttc ata gaa aac ggc tgg gaa gga 1109 Gly Ile Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly 345 350 355 360 atg gtt gat ggg tgg tat gga ttc cga tat caa aac tcg gaa gga aca 1157 Met Val Asp Gly Trp Tyr Gly Phe Arg Tyr Gln Asn Ser Glu Gly Thr 365 370 375 gga caa gct gca gat cta aag agc act caa gca gcc atc gac cag atc 1205 Gly Gln Ala Ala Asp Leu Lys Ser Thr Gln Ala Ala Ile Asp Gln Ile 380 385 390 aat gga aaa tta aac aga gtg att gaa agg acc aat gag aaa ttc cat 1253 Asn Gly Lys Leu Asn Arg Val Ile Glu Arg Thr Asn Glu Lys Phe His 395 400 405 caa ata gag aag gaa ttc tca gaa gta gaa ggg agg atc cag gac ttg 1301 Gln Ile Glu Lys Glu Phe Ser Glu Val Glu Gly Arg Ile Gln Asp Leu 410 415 420 gag aag tat gta gaa gac acc aaa ata gac cta tgg tcc tac aat gca 1349 Glu Lys Tyr Val Glu Asp Thr Lys Ile Asp Leu Trp Ser Tyr Asn Ala 425 430 435 440 gaa ttg ctg gtg gct cta aaa aat caa cat aca att gac tta aca gat 1397 Glu Leu Leu Val Ala Leu Lys Asn Gln His Thr Ile Asp Leu Thr Asp 445 450 455 gca gaa atg aat aaa tta ttc gag aag act aga cgc cag tta aga gaa 1445 Ala Glu Met Asn Lys Leu Phe Glu Lys Thr Arg Arg Gln Leu Arg Glu 460 465 470 aac gcg gaa gac atg gga ggt gga tgt ttc aag ata tac cac aaa tgt 1493 Asn Ala Glu Asp Met Gly Gly Gly Cys Phe Lys Ile Tyr His Lys Cys 475 480 485 gat aat gca tgc att gga tca ata aga aat ggg aca tat gac cat tac 1541 Asp Asn Ala Cys Ile Gly Ser Ile Arg Asn Gly Thr Tyr Asp His Tyr 490 495 500 ata tac aga gat gaa gca tta aac aac cgg ttt caa atc aaa ggt gtt 1589 Ile Tyr Arg Asp Glu Ala Leu Asn Asn Arg Phe Gln Ile Lys Gly Val 505 510 515 520 gag ttg aaa tca ggc tac aaa gat tgg ata ctg tgg att tca ttc gcc 1637 Glu Leu Lys Ser Gly Tyr Lys Asp Trp Ile Leu Trp Ile Ser Phe Ala 525 530 535 ata tca tgc ttc tta att tgc gtt gtt cta ttg ggt ttc att atg tgg 1685 Ile Ser Cys Phe Leu Ile Cys Val Val Leu Leu Gly Phe Ile Met Trp 540 545 550 gct tgc caa aaa ggc aac atc aga tgc aac att tgc att tgagtaaact 1734 Ala Cys Gln Lys Gly Asn Ile Arg Cys Asn Ile Cys Ile 555 560 565 gatagttaaa aacacccttg tttctact 1762 8 565 PRT Equine influenza virus H3N8 8 Met Lys Thr Thr Ile Ile Leu Ile Pro Leu Thr His Trp Val Tyr Ser 1 5 10 15 Gln Asn Pro Thr Ser Gly Asn Asn Thr Ala Thr Leu Cys Leu Gly His 20 25 30 His Ala Val Ala Asn Gly Thr Leu Val Lys Thr Ile Thr Asp Asp Gln 35 40 45 Ile Glu Val Thr Asn Ala Thr Glu Leu Val Gln Ser Ile Ser Ile Gly 50 55 60 Lys Ile Cys Asn Asn Ser Tyr Arg Val Leu Asp Gly Arg Asn Cys Thr 65 70 75 80 Leu Ile Asp Ala Met Leu Gly Asp Pro His Cys Asp Val Phe Gln Tyr 85 90 95 Glu Asn Trp Asp Leu Phe Ile Glu Arg Ser Ser Ala Phe Ser Ser Cys 100 105 110 Tyr Pro Tyr Asp Ile Pro Asp Tyr Ala Ser Leu Arg Ser Ile Val Ala 115 120 125 Ser Ser Gly Thr Leu Glu Phe Thr Ala Glu Gly Phe Thr Trp Thr Gly 130 135 140 Val Thr Gln Asn Gly Arg Ser Gly Ser Cys Lys Arg Gly Ser Ala Asp 145 150 155 160 Ser Phe Phe Ser Arg Leu Asn Trp Leu Thr Glu Ser Gly Asn Ser Tyr 165 170 175 Pro Thr Leu Asn Val Thr Met Pro Asn Asn Lys Asn Phe Asp Lys Leu 180 185 190 Tyr Ile Trp Gly Ile His His Pro Ser Ser Asn Lys Glu Gln Thr Lys 195 200 205 Leu Tyr Ile Gln Glu Ser Gly Arg Val Thr Val Ser Thr Lys Arg Ser 210 215 220 Gln Gln Thr Ile Ile Pro Asn Ile Gly Ser Arg Pro Arg Val Arg Gly 225 230 235 240 Gln Ser Gly Arg Ile Ser Ile Tyr Trp Thr Ile Val Lys Pro Gly Asp 245 250 255 Ile Leu Met Ile Asn Ser Asn Gly Asn Leu Val Ala Pro Arg Gly Tyr 260 265 270 Phe Lys Leu Lys Thr Gly Lys Ser Ser Val Met Arg Ser Asp Ala Pro 275 280 285 Ile Asp Ile Cys Val Ser Glu Cys Ile Thr Pro Asn Gly Ser Ile Pro 290 295 300 Asn Asp Lys Pro Phe Gln Asn Val Asn Lys Val Thr Tyr Gly Lys Cys 305 310 315 320 Pro Lys Tyr Ile Arg Gln Asn Thr Leu Lys Leu Ala Thr Gly Met Arg 325 330 335 Asn Val Pro Glu Lys Gln Ile Arg Gly Ile Phe Gly Ala Ile Ala Gly 340 345 350 Phe Ile Glu Asn Gly Trp Glu Gly Met Val Asp Gly Trp Tyr Gly Phe 355 360 365 Arg Tyr Gln Asn Ser Glu Gly Thr Gly Gln Ala Ala Asp Leu Lys Ser 370 375 380 Thr Gln Ala Ala Ile Asp Gln Ile Asn Gly Lys Leu Asn Arg Val Ile 385 390 395 400 Glu Arg Thr Asn Glu Lys Phe His Gln Ile Glu Lys Glu Phe Ser Glu 405 410 415 Val Glu Gly Arg Ile Gln Asp Leu Glu Lys Tyr Val Glu Asp Thr Lys 420 425 430 Ile Asp Leu Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Lys Asn 435 440 445 Gln His Thr Ile Asp Leu Thr Asp Ala Glu Met Asn Lys Leu Phe Glu 450 455 460 Lys Thr Arg Arg Gln Leu Arg Glu Asn Ala Glu Asp Met Gly Gly Gly 465 470 475 480 Cys Phe Lys Ile Tyr His Lys Cys Asp Asn Ala Cys Ile Gly Ser Ile 485 490 495 Arg Asn Gly Thr Tyr Asp His Tyr Ile Tyr Arg Asp Glu Ala Leu Asn 500 505 510 Asn Arg Phe Gln Ile Lys Gly Val Glu Leu Lys Ser Gly Tyr Lys Asp 515 520 525 Trp Ile Leu Trp Ile Ser Phe Ala Ile Ser Cys Phe Leu Ile Cys Val 530 535 540 Val Leu Leu Gly Phe Ile Met Trp Ala Cys Gln Lys Gly Asn Ile Arg 545 550 555 560 Cys Asn Ile Cys Ile 565 9 1695 DNA Equine influenza virus H3N8 9 atgaagacaa ccattatttt gataccactg acccattggg tctacagtca aaacccaacc 60 agtggcaaca acacagccac attatgtctg ggacaccatg cagtagcaaa tggaacattg 120 gtaaaaacaa taactgatga ccaaattgag gtgacaaatg ctactgaatt agttcagagc 180 atttcaatag ggaaaatatg caacaactca tatagagttc tagatggaag aaattgcaca 240 ttaatagatg caatgctagg agacccccac tgtgatgtct ttcagtatga gaattgggac 300 ctcttcatag aaagaagcag cgctttcagc agttgctacc catatgacat ccctgactat 360 gcatcgctcc ggtccattgt agcatcctca ggaacattgg aattcacagc agagggattc 420 acatggacag gtgtcactca aaacggaaga agtggatcct gcaaaagggg atcagccgat 480 agtttcttta gccgactgaa ttggctaaca gaatctggaa actcttaccc cacattgaat 540 gtgacaatgc ctaacaataa aaatttcgac aaactataca tctgggggat tcatcacccg 600 agctcaaaca aagagcagac aaaattgtac atccaagaat cgggacgagt aacagtctca 660 acaaaaagaa gtcaacaaac aataatccct aacatcggat ctagaccgcg ggtcaggggt 720 caatcaggca ggataagcat atactggacc attgtaaaac ctggagatat cctaatgata 780 aacagtaatg gcaacttagt tgcaccgcgg ggatatttta aattgaaaac agggaaaagc 840 tctgtaatga gatcagatgc acccatagac atttgtgtgt ctgaatgtat tacaccaaat 900 ggaagcatcc ccaacgacaa accatttcaa aatgtgaaca aagttacata tggaaaatgc 960 cccaagtata tcaggcaaaa cactttaaag ctggccactg ggatgaggaa tgtaccagaa 1020 aagcaaatca gaggaatctt tggagcaata gcgggattca tagaaaacgg ctgggaagga 1080 atggttgatg ggtggtatgg attccgatat caaaactcgg aaggaacagg acaagctgca 1140 gatctaaaga gcactcaagc agccatcgac cagatcaatg gaaaattaaa cagagtgatt 1200 gaaaggacca atgagaaatt ccatcaaata gagaaggaat tctcagaagt agaagggagg 1260 atccaggact tggagaagta tgtagaagac accaaaatag acctatggtc ctacaatgca 1320 gaattgctgg tggctctaaa aaatcaacat acaattgact taacagatgc agaaatgaat 1380 aaattattcg agaagactag acgccagtta agagaaaacg cggaagacat gggaggtgga 1440 tgtttcaaga tataccacaa atgtgataat gcatgcattg gatcaataag aaatgggaca 1500 tatgaccatt acatatacag agatgaagca ttaaacaacc ggtttcaaat caaaggtgtt 1560 gagttgaaat caggctacaa agattggata ctgtggattt cattcgccat atcatgcttc 1620 ttaatttgcg ttgttctatt gggtttcatt atgtgggctt gccaaaaagg caacatcaga 1680 tgcaacattt gcatt 1695 10 1762 DNA Equine influenza virus H3N8 CDS (30)..(1724) 10 agcaaaagca ggggatattt ctgtcaatc atg aag aca acc att att ttg ata 53 Met Lys Thr Thr Ile Ile Leu Ile 1 5 cta ctg acc cat tgg gtc tac agt caa aac cca acc agt ggc aac aac 101 Leu Leu Thr His Trp Val Tyr Ser Gln Asn Pro Thr Ser Gly Asn Asn 10 15 20 aca gcc aca tta tgt ctg gga cac cat gca gta gca aat gga aca ttg 149 Thr Ala Thr Leu Cys Leu Gly His His Ala Val Ala Asn Gly Thr Leu 25 30 35 40 gta aaa aca ata act gat gac caa att gag gtg aca aat gct act gaa 197 Val Lys Thr Ile Thr Asp Asp Gln Ile Glu Val Thr Asn Ala Thr Glu 45 50 55 tta gtt cag agc att tca ata ggg aaa ata tgc aac aac tca tat aga 245 Leu Val Gln Ser Ile Ser Ile Gly Lys Ile Cys Asn Asn Ser Tyr Arg 60 65 70 gtt cta gat gga aga aat tgc aca tta ata gat gca atg cta gga gac 293 Val Leu Asp Gly Arg Asn Cys Thr Leu Ile Asp Ala Met Leu Gly Asp 75 80 85 ccc cac tgt gat gtc ttt cag tat gag aat tgg gac ctc ttc ata gaa 341 Pro His Cys Asp Val Phe Gln Tyr Glu Asn Trp Asp Leu Phe Ile Glu 90 95 100 aga agc agc gct ttc agc agt tgc tac cca tat gac atc cct gac tat 389 Arg Ser Ser Ala Phe Ser Ser Cys Tyr Pro Tyr Asp Ile Pro Asp Tyr 105 110 115 120 gca tcg ctc cgg tcc att gta gca tcc tca gga aca ttg gaa ttc aca 437 Ala Ser Leu Arg Ser Ile Val Ala Ser Ser Gly Thr Leu Glu Phe Thr 125 130 135 gca gag gga ttc aca tgg aca ggt gtc act caa aac gga aga agt gga 485 Ala Glu Gly Phe Thr Trp Thr Gly Val Thr Gln Asn Gly Arg Ser Gly 140 145 150 tcc tgc aaa agg gaa tca gcc gat agt ttc ttt agc cga ctg aat tgg 533 Ser Cys Lys Arg Glu Ser Ala Asp Ser Phe Phe Ser Arg Leu Asn Trp 155 160 165 cta aca gaa tct gga aac tct tac ccc aca ttg aat gtg aca atg cct 581 Leu Thr Glu Ser Gly Asn Ser Tyr Pro Thr Leu Asn Val Thr Met Pro 170 175 180 aac aat aaa aat ttc gac aaa cta tac atc tgg ggg att cat cac ccg 629 Asn Asn Lys Asn Phe Asp Lys Leu Tyr Ile Trp Gly Ile His His Pro 185 190 195 200 agc tca aac aaa gag cag aca aaa ttg tac atc caa gaa tca gga cga 677 Ser Ser Asn Lys Glu Gln Thr Lys Leu Tyr Ile Gln Glu Ser Gly Arg 205 210 215 gta aca gtc tca aca aaa aga agt caa caa aca ata atc cct aac atc 725 Val Thr Val Ser Thr Lys Arg Ser Gln Gln Thr Ile Ile Pro Asn Ile 220 225 230 gga tct aga ccg tgg gtc agg ggt caa tca ggc agg ata agc ata tac 773 Gly Ser Arg Pro Trp Val Arg Gly Gln Ser Gly Arg Ile Ser Ile Tyr 235 240 245 tgg acc att gta aaa cct gga gat atc cta acg ata aac agt aat ggc 821 Trp Thr Ile Val Lys Pro Gly Asp Ile Leu Thr Ile Asn Ser Asn Gly 250 255 260 aac tta gtt gca ccg cgg gga tat ttt aaa ttg aaa aca ggg aaa agc 869 Asn Leu Val Ala Pro Arg Gly Tyr Phe Lys Leu Lys Thr Gly Lys Ser 265 270 275 280 tct gta atg aga tca gat gca ccc ata gac att tgt gtg tct gaa tgt 917 Ser Val Met Arg Ser Asp Ala Pro Ile Asp Ile Cys Val Ser Glu Cys 285 290 295 att aca cca aat gga agc atc ccc aac gac aaa cca ttt caa aat gtg 965 Ile Thr Pro Asn Gly Ser Ile Pro Asn Asp Lys Pro Phe Gln Asn Val 300 305 310 aac aaa gtt aca tat gga aaa tgc ccc aag tat atc agg caa aac act 1013 Asn Lys Val Thr Tyr Gly Lys Cys Pro Lys Tyr Ile Arg Gln Asn Thr 315 320 325 tta aag ctg gcc act ggg atg agg aat gta cca gaa aag caa atc aga 1061 Leu Lys Leu Ala Thr Gly Met Arg Asn Val Pro Glu Lys Gln Ile Arg 330 335 340 gga atc ttt gga gca ata gcg gga ttc ata gaa aac ggc tgg gaa gga 1109 Gly Ile Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly 345 350 355 360 atg gtt gat ggg tgg tat gga ttc cga tat caa aac tcg gaa gga aca 1157 Met Val Asp Gly Trp Tyr Gly Phe Arg Tyr Gln Asn Ser Glu Gly Thr 365 370 375 gga caa gct gca gat cta aag agc act caa gca gcc atc gac cag atc 1205 Gly Gln Ala Ala Asp Leu Lys Ser Thr Gln Ala Ala Ile Asp Gln Ile 380 385 390 aat gga aaa tta aac aga gtg att gaa agg acc aat gag aaa ttc cat 1253 Asn Gly Lys Leu Asn Arg Val Ile Glu Arg Thr Asn Glu Lys Phe His 395 400 405 caa ata gag aag gaa ttc tca gaa gta gaa ggg aga atc cag gac ttg 1301 Gln Ile Glu Lys Glu Phe Ser Glu Val Glu Gly Arg Ile Gln Asp Leu 410 415 420 gag aag tat gta gaa gac acc aaa ata gac cta tgg tcc tac aat gca 1349 Glu Lys Tyr Val Glu Asp Thr Lys Ile Asp Leu Trp Ser Tyr Asn Ala 425 430 435 440 gaa ttg ctg gtg gct cta gaa aat caa cat aca att gac tta aca gat 1397 Glu Leu Leu Val Ala Leu Glu Asn Gln His Thr Ile Asp Leu Thr Asp 445 450 455 gca gaa atg aat aaa tta ttc gag aag act aga cgc cag tta aga gaa 1445 Ala Glu Met Asn Lys Leu Phe Glu Lys Thr Arg Arg Gln Leu Arg Glu 460 465 470 aac gcg gaa gac atg gga ggt gga tgt ttc aag ata tac cac aaa tgt 1493 Asn Ala Glu Asp Met Gly Gly Gly Cys Phe Lys Ile Tyr His Lys Cys 475 480 485 gat aat gca tgc att gga tca ata aga aat ggg aca tat gac cat tac 1541 Asp Asn Ala Cys Ile Gly Ser Ile Arg Asn Gly Thr Tyr Asp His Tyr 490 495 500 ata tac aga gat gaa gca tta aac aac cgg ttt caa atc aaa ggt gtt 1589 Ile Tyr Arg Asp Glu Ala Leu Asn Asn Arg Phe Gln Ile Lys Gly Val 505 510 515 520 gag ttg aaa tca ggc tac aaa gat tgg ata ctg tgg att tca ttc gcc 1637 Glu Leu Lys Ser Gly Tyr Lys Asp Trp Ile Leu Trp Ile Ser Phe Ala 525 530 535 ata tca tgc ttc tta att tgc gtt gtt cta ttg ggt ttc att atg tgg 1685 Ile Ser Cys Phe Leu Ile Cys Val Val Leu Leu Gly Phe Ile Met Trp 540 545 550 gct tgc caa aaa ggc aac atc aga tgc aac att tgc att tgagtaaact 1734 Ala Cys Gln Lys Gly Asn Ile Arg Cys Asn Ile Cys Ile 555 560 565 gatagttaaa aacacccttg tttctact 1762 11 565 PRT Equine influenza virus H3N8 11 Met Lys Thr Thr Ile Ile Leu Ile Leu Leu Thr His Trp Val Tyr Ser 1 5 10 15 Gln Asn Pro Thr Ser Gly Asn Asn Thr Ala Thr Leu Cys Leu Gly His 20 25 30 His Ala Val Ala Asn Gly Thr Leu Val Lys Thr Ile Thr Asp Asp Gln 35 40 45 Ile Glu Val Thr Asn Ala Thr Glu Leu Val Gln Ser Ile Ser Ile Gly 50 55 60 Lys Ile Cys Asn Asn Ser Tyr Arg Val Leu Asp Gly Arg Asn Cys Thr 65 70 75 80 Leu Ile Asp Ala Met Leu Gly Asp Pro His Cys Asp Val Phe Gln Tyr 85 90 95 Glu Asn Trp Asp Leu Phe Ile Glu Arg Ser Ser Ala Phe Ser Ser Cys 100 105 110 Tyr Pro Tyr Asp Ile Pro Asp Tyr Ala Ser Leu Arg Ser Ile Val Ala 115 120 125 Ser Ser Gly Thr Leu Glu Phe Thr Ala Glu Gly Phe Thr Trp Thr Gly 130 135 140 Val Thr Gln Asn Gly Arg Ser Gly Ser Cys Lys Arg Glu Ser Ala Asp 145 150 155 160 Ser Phe Phe Ser Arg Leu Asn Trp Leu Thr Glu Ser Gly Asn Ser Tyr 165 170 175 Pro Thr Leu Asn Val Thr Met Pro Asn Asn Lys Asn Phe Asp Lys Leu 180 185 190 Tyr Ile Trp Gly Ile His His Pro Ser Ser Asn Lys Glu Gln Thr Lys 195 200 205 Leu Tyr Ile Gln Glu Ser Gly Arg Val Thr Val Ser Thr Lys Arg Ser 210 215 220 Gln Gln Thr Ile Ile Pro Asn Ile Gly Ser Arg Pro Trp Val Arg Gly 225 230 235 240 Gln Ser Gly Arg Ile Ser Ile Tyr Trp Thr Ile Val Lys Pro Gly Asp 245 250 255 Ile Leu Thr Ile Asn Ser Asn Gly Asn Leu Val Ala Pro Arg Gly Tyr 260 265 270 Phe Lys Leu Lys Thr Gly Lys Ser Ser Val Met Arg Ser Asp Ala Pro 275 280 285 Ile Asp Ile Cys Val Ser Glu Cys Ile Thr Pro Asn Gly Ser Ile Pro 290 295 300 Asn Asp Lys Pro Phe Gln Asn Val Asn Lys Val Thr Tyr Gly Lys Cys 305 310 315 320 Pro Lys Tyr Ile Arg Gln Asn Thr Leu Lys Leu Ala Thr Gly Met Arg 325 330 335 Asn Val Pro Glu Lys Gln Ile Arg Gly Ile Phe Gly Ala Ile Ala Gly 340 345 350 Phe Ile Glu Asn Gly Trp Glu Gly Met Val Asp Gly Trp Tyr Gly Phe 355 360 365 Arg Tyr Gln Asn Ser Glu Gly Thr Gly Gln Ala Ala Asp Leu Lys Ser 370 375 380 Thr Gln Ala Ala Ile Asp Gln Ile Asn Gly Lys Leu Asn Arg Val Ile 385 390 395 400 Glu Arg Thr Asn Glu Lys Phe His Gln Ile Glu Lys Glu Phe Ser Glu 405 410 415 Val Glu Gly Arg Ile Gln Asp Leu Glu Lys Tyr Val Glu Asp Thr Lys 420 425 430 Ile Asp Leu Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu Asn 435 440 445 Gln His Thr Ile Asp Leu Thr Asp Ala Glu Met Asn Lys Leu Phe Glu 450 455 460 Lys Thr Arg Arg Gln Leu Arg Glu Asn Ala Glu Asp Met Gly Gly Gly 465 470 475 480 Cys Phe Lys Ile Tyr His Lys Cys Asp Asn Ala Cys Ile Gly Ser Ile 485 490 495 Arg Asn Gly Thr Tyr Asp His Tyr Ile Tyr Arg Asp Glu Ala Leu Asn 500 505 510 Asn Arg Phe Gln Ile Lys Gly Val Glu Leu Lys Ser Gly Tyr Lys Asp 515 520 525 Trp Ile Leu Trp Ile Ser Phe Ala Ile Ser Cys Phe Leu Ile Cys Val 530 535 540 Val Leu Leu Gly Phe Ile Met Trp Ala Cys Gln Lys Gly Asn Ile Arg 545 550 555 560 Cys Asn Ile Cys Ile 565 12 1695 DNA Equine influenza virus H3N8 12 atgaagacaa ccattatttt gatactactg acccattggg tctacagtca aaacccaacc 60 agtggcaaca acacagccac attatgtctg ggacaccatg cagtagcaaa tggaacattg 120 gtaaaaacaa taactgatga ccaaattgag gtgacaaatg ctactgaatt agttcagagc 180 atttcaatag ggaaaatatg caacaactca tatagagttc tagatggaag aaattgcaca 240 ttaatagatg caatgctagg agacccccac tgtgatgtct ttcagtatga gaattgggac 300 ctcttcatag aaagaagcag cgctttcagc agttgctacc catatgacat ccctgactat 360 gcatcgctcc ggtccattgt agcatcctca ggaacattgg aattcacagc agagggattc 420 acatggacag gtgtcactca aaacggaaga agtggatcct gcaaaaggga atcagccgat 480 agtttcttta gccgactgaa ttggctaaca gaatctggaa actcttaccc cacattgaat 540 gtgacaatgc ctaacaataa aaatttcgac aaactataca tctgggggat tcatcacccg 600 agctcaaaca aagagcagac aaaattgtac atccaagaat caggacgagt aacagtctca 660 acaaaaagaa gtcaacaaac aataatccct aacatcggat ctagaccgtg ggtcaggggt 720 caatcaggca ggataagcat atactggacc attgtaaaac ctggagatat cctaacgata 780 aacagtaatg gcaacttagt tgcaccgcgg ggatatttta aattgaaaac agggaaaagc 840 tctgtaatga gatcagatgc acccatagac atttgtgtgt ctgaatgtat tacaccaaat 900 ggaagcatcc ccaacgacaa accatttcaa aatgtgaaca aagttacata tggaaaatgc 960 cccaagtata tcaggcaaaa cactttaaag ctggccactg ggatgaggaa tgtaccagaa 1020 aagcaaatca gaggaatctt tggagcaata gcgggattca tagaaaacgg ctgggaagga 1080 atggttgatg ggtggtatgg attccgatat caaaactcgg aaggaacagg acaagctgca 1140 gatctaaaga gcactcaagc agccatcgac cagatcaatg gaaaattaaa cagagtgatt 1200 gaaaggacca atgagaaatt ccatcaaata gagaaggaat tctcagaagt agaagggaga 1260 atccaggact tggagaagta tgtagaagac accaaaatag acctatggtc ctacaatgca 1320 gaattgctgg tggctctaga aaatcaacat acaattgact taacagatgc agaaatgaat 1380 aaattattcg agaagactag acgccagtta agagaaaacg cggaagacat gggaggtgga 1440 tgtttcaaga tataccacaa atgtgataat gcatgcattg gatcaataag aaatgggaca 1500 tatgaccatt acatatacag agatgaagca ttaaacaacc ggtttcaaat caaaggtgtt 1560 gagttgaaat caggctacaa agattggata ctgtggattt cattcgccat atcatgcttc 1620 ttaatttgcg ttgttctatt gggtttcatt atgtgggctt gccaaaaagg caacatcaga 1680 tgcaacattt gcatt 1695 13 1241 DNA Equine influenza virus H3N8 CDS (28)..(1239) 13 agcaaaagca ggtcaaatat attcaat atg gag aga ata aaa gaa ctg aga gat 54 Met Glu Arg Ile Lys Glu Leu Arg Asp 1 5 cta atg tca caa tcc cgc acc cgc gag ata cta aca aaa act act gtg 102 Leu Met Ser Gln Ser Arg Thr Arg Glu Ile Leu Thr Lys Thr Thr Val 10 15 20 25 gac cac atg gcc ata atc aag aaa tac aca tca gga aga caa gag aag 150 Asp His Met Ala Ile Ile Lys Lys Tyr Thr Ser Gly Arg Gln Glu Lys 30 35 40 aac ccc gca ctt agg atg aag tgg atg atg gca atg aaa tac cca att 198 Asn Pro Ala Leu Arg Met Lys Trp Met Met Ala Met Lys Tyr Pro Ile 45 50 55 aca gca gat aag agg ata atg gaa atg att cct gag aga aat gaa cag 246 Thr Ala Asp Lys Arg Ile Met Glu Met Ile Pro Glu Arg Asn Glu Gln 60 65 70 ggg caa acc ctt tgg agc aaa acg aac gat gct ggc tca gac cgc gta 294 Gly Gln Thr Leu Trp Ser Lys Thr Asn Asp Ala Gly Ser Asp Arg Val 75 80 85 atg gta tca cct ctg gca gtg aca tgg tgg aat agg aat gga cca aca 342 Met Val Ser Pro Leu Ala Val Thr Trp Trp Asn Arg Asn Gly Pro Thr 90 95 100 105 acg agc aca att cat tat cca aaa gtc tac aaa act tat ttt gaa aaa 390 Thr Ser Thr Ile His Tyr Pro Lys Val Tyr Lys Thr Tyr Phe Glu Lys 110 115 120 gtt gaa aga tta aaa cac gga acc ttt ggc ccc gtt cat ttt agg aat 438 Val Glu Arg Leu Lys His Gly Thr Phe Gly Pro Val His Phe Arg Asn 125 130 135 caa gtc aag ata aga cgg aga gtt gat gta aac cct ggt cac gcg gac 486 Gln Val Lys Ile Arg Arg Arg Val Asp Val Asn Pro Gly His Ala Asp 140 145 150 ctc agt gcc aaa gaa gca caa gat gtg atc atg gaa gtt gtt ttc cca 534 Leu Ser Ala Lys Glu Ala Gln Asp Val Ile Met Glu Val Val Phe Pro 155 160 165 aat gaa gtg gga gcc aga att cta aca tcg gaa tca caa cta aca ata 582 Asn Glu Val Gly Ala Arg Ile Leu Thr Ser Glu Ser Gln Leu Thr Ile 170 175 180 185 acc aaa gag aaa aaa gaa gaa ctt cag gac tgc aaa att gcc ccc ttg 630 Thr Lys Glu Lys Lys Glu Glu Leu Gln Asp Cys Lys Ile Ala Pro Leu 190 195 200 atg gta gca tac atg cta gaa aga gag ttg gtc cga aaa aca aga ttc 678 Met Val Ala Tyr Met Leu Glu Arg Glu Leu Val Arg Lys Thr Arg Phe 205 210 215 ctc cca gtg gct ggc gga aca agc agt gta tac att gaa gtg ttg cat 726 Leu Pro Val Ala Gly Gly Thr Ser Ser Val Tyr Ile Glu Val Leu His 220 225 230 ctg act cag gga aca tgc tgg gaa caa atg tac acc cca gga gga gaa 774 Leu Thr Gln Gly Thr Cys Trp Glu Gln Met Tyr Thr Pro Gly Gly Glu 235 240 245 gtt aga aac gat gac att gat caa agt tta att att gct gcc cgg aac 822 Val Arg Asn Asp Asp Ile Asp Gln Ser Leu Ile Ile Ala Ala Arg Asn 250 255 260 265 ata gtg aga aga gcg aca gta tca gca gat cca cta gca tcc ctg ctg 870 Ile Val Arg Arg Ala Thr Val Ser Ala Asp Pro Leu Ala Ser Leu Leu 270 275 280 gaa atg tgc cac agt aca cag att ggt gga ata agg atg gta gac atc 918 Glu Met Cys His Ser Thr Gln Ile Gly Gly Ile Arg Met Val Asp Ile 285 290 295 ctt aag cag aat cca aca gag gaa caa gct gtg gat ata tgc aaa gca 966 Leu Lys Gln Asn Pro Thr Glu Glu Gln Ala Val Asp Ile Cys Lys Ala 300 305 310 gca atg ggg tta aga att agc tca tca ttc agc ttt ggt gga ttc acc 1014 Ala Met Gly Leu Arg Ile Ser Ser Ser Phe Ser Phe Gly Gly Phe Thr 315 320 325 ttt aag aga aca agt gga tca tca gtc aag aga gaa gaa gaa atg ctt 1062 Phe Lys Arg Thr Ser Gly Ser Ser Val Lys Arg Glu Glu Glu Met Leu 330 335 340 345 acg ggc aac ctt caa aca ttg aaa ata aga gtg cat gaa ggc tat gaa 1110 Thr Gly Asn Leu Gln Thr Leu Lys Ile Arg Val His Glu Gly Tyr Glu 350 355 360 gaa ttc aca atg gtc gga aga aga gca aca gcc att ctc aga aag gca 1158 Glu Phe Thr Met Val Gly Arg Arg Ala Thr Ala Ile Leu Arg Lys Ala 365 370 375 acc aga aga ttg att caa ttg ata gta agt ggg aga gat gaa caa tca 1206 Thr Arg Arg Leu Ile Gln Leu Ile Val Ser Gly Arg Asp Glu Gln Ser 380 385 390 att gct gaa gca ata att gta gcc atg gtg ttt tc 1241 Ile Ala Glu Ala Ile Ile Val Ala Met Val Phe 395 400 14 404 PRT Equine influenza virus H3N8 14 Met Glu Arg Ile Lys Glu Leu Arg Asp Leu Met Ser Gln Ser Arg Thr 1 5 10 15 Arg Glu Ile Leu Thr Lys Thr Thr Val Asp His Met Ala Ile Ile Lys 20 25 30 Lys Tyr Thr Ser Gly Arg Gln Glu Lys Asn Pro Ala Leu Arg Met Lys 35 40 45 Trp Met Met Ala Met Lys Tyr Pro Ile Thr Ala Asp Lys Arg Ile Met 50 55 60 Glu Met Ile Pro Glu Arg Asn Glu Gln Gly Gln Thr Leu Trp Ser Lys 65 70 75 80 Thr Asn Asp Ala Gly Ser Asp Arg Val Met Val Ser Pro Leu Ala Val 85 90 95 Thr Trp Trp Asn Arg Asn Gly Pro Thr Thr Ser Thr Ile His Tyr Pro 100 105 110 Lys Val Tyr Lys Thr Tyr Phe Glu Lys Val Glu Arg Leu Lys His Gly 115 120 125 Thr Phe Gly Pro Val His Phe Arg Asn Gln Val Lys Ile Arg Arg Arg 130 135 140 Val Asp Val Asn Pro Gly His Ala Asp Leu Ser Ala Lys Glu Ala Gln 145 150 155 160 Asp Val Ile Met Glu Val Val Phe Pro Asn Glu Val Gly Ala Arg Ile 165 170 175 Leu Thr Ser Glu Ser Gln Leu Thr Ile Thr Lys Glu Lys Lys Glu Glu 180 185 190 Leu Gln Asp Cys Lys Ile Ala Pro Leu Met Val Ala Tyr Met Leu Glu 195 200 205 Arg Glu Leu Val Arg Lys Thr Arg Phe Leu Pro Val Ala Gly Gly Thr 210 215 220 Ser Ser Val Tyr Ile Glu Val Leu His Leu Thr Gln Gly Thr Cys Trp 225 230 235 240 Glu Gln Met Tyr Thr Pro Gly Gly Glu Val Arg Asn Asp Asp Ile Asp 245 250 255 Gln Ser Leu Ile Ile Ala Ala Arg Asn Ile Val Arg Arg Ala Thr Val 260 265 270 Ser Ala Asp Pro Leu Ala Ser Leu Leu Glu Met Cys His Ser Thr Gln 275 280 285 Ile Gly Gly Ile Arg Met Val Asp Ile Leu Lys Gln Asn Pro Thr Glu 290 295 300 Glu Gln Ala Val Asp Ile Cys Lys Ala Ala Met Gly Leu Arg Ile Ser 305 310 315 320 Ser Ser Phe Ser Phe Gly Gly Phe Thr Phe Lys Arg Thr Ser Gly Ser 325 330 335 Ser Val Lys Arg Glu Glu Glu Met Leu Thr Gly Asn Leu Gln Thr Leu 340 345 350 Lys Ile Arg Val His Glu Gly Tyr Glu Glu Phe Thr Met Val Gly Arg 355 360 365 Arg Ala Thr Ala Ile Leu Arg Lys Ala Thr Arg Arg Leu Ile Gln Leu 370 375 380 Ile Val Ser Gly Arg Asp Glu Gln Ser Ile Ala Glu Ala Ile Ile Val 385 390 395 400 Ala Met Val Phe 15 1214 DNA Equine influenza virus H3N8 15 atggagagaa taaaagaact gagagatcta atgtcacaat cccgcacccg cgagatacta 60 acaaaaacta ctgtggacca catggccata atcaagaaat acacatcagg aagacaagag 120 aagaaccccg cacttaggat gaagtggatg atggcaatga aatacccaat tacagcagat 180 aagaggataa tggaaatgat tcctgagaga aatgaacagg ggcaaaccct ttggagcaaa 240 acgaacgatg ctggctcaga ccgcgtaatg gtatcacctc tggcagtgac atggtggaat 300 aggaatggac caacaacgag cacaattcat tatccaaaag tctacaaaac ttattttgaa 360 aaagttgaaa gattaaaaca cggaaccttt ggccccgttc attttaggaa tcaagtcaag 420 ataagacgga gagttgatgt aaaccctggt cacgcggacc tcagtgccaa agaagcacaa 480 gatgtgatca tggaagttgt tttcccaaat gaagtgggag ccagaattct aacatcggaa 540 tcacaactaa caataaccaa agagaaaaaa gaagaacttc aggactgcaa aattgccccc 600 ttgatggtag catacatgct agaaagagag ttggtccgaa aaacaagatt cctcccagtg 660 gctggcggaa caagcagtgt atacattgaa gtgttgcatc tgactcaggg aacatgctgg 720 gaacaaatgt acaccccagg aggagaagtt agaaacgatg acattgatca aagtttaatt 780 attgctgccc ggaacatagt gagaagagcg acagtatcag cagatccact agcatccctg 840 ctggaaatgt gccacagtac acagattggt ggaataagga tggtagacat ccttaagcag 900 aatccaacag aggaacaagc tgtggatata tgcaaagcag caatggggtt aagaattagc 960 tcatcattca gctttggtgg attcaccttt aagagaacaa gtggatcatc agtcaagaga 1020 gaagaagaaa tgcttacggg caaccttcaa acattgaaaa taagagtgca tgaaggctat 1080 gaagaattca caatggtcgg aagaagagca acagccattc tcagaaaggc aaccagaaga 1140 ttgattcaat tgatagtaag tgggagagat gaacaatcaa ttgctgaagc aataattgta 1200 gccatggtgt tttc 1214 16 1241 DNA Equine influenza virus H3N8 CDS (28)..(1239) 16 agcaaaagca ggtcaaatat attcaat atg gag aga ata aaa gaa ctg aga gat 54 Met Glu Arg Ile Lys Glu Leu Arg Asp 1 5 cta atg tca caa tcc cgc acc cgc gag ata cta aca aaa act act gtg 102 Leu Met Ser Gln Ser Arg Thr Arg Glu Ile Leu Thr Lys Thr Thr Val 10 15 20 25 gac cac atg gcc ata atc aag aaa tac aca tca gga aga caa gag aag 150 Asp His Met Ala Ile Ile Lys Lys Tyr Thr Ser Gly Arg Gln Glu Lys 30 35 40 aac ccc gca ctt agg atg aag tgg atg atg gca atg aaa tac cca att 198 Asn Pro Ala Leu Arg Met Lys Trp Met Met Ala Met Lys Tyr Pro Ile 45 50 55 aca gca gat aag agg ata atg gaa atg att cct gag aga aat gaa cag 246 Thr Ala Asp Lys Arg Ile Met Glu Met Ile Pro Glu Arg Asn Glu Gln 60 65 70 ggg caa acc ctt tgg agc aaa acg aac gat gct ggc tca gac cgc gta 294 Gly Gln Thr Leu Trp Ser Lys Thr Asn Asp Ala Gly Ser Asp Arg Val 75 80 85 atg gta tca cct ctg gca gtg aca tgg tgg aat agg aat gga cca aca 342 Met Val Ser Pro Leu Ala Val Thr Trp Trp Asn Arg Asn Gly Pro Thr 90 95 100 105 acg agc aca att cat tat cca aaa gtc cac aaa act tat ttt gaa aaa 390 Thr Ser Thr Ile His Tyr Pro Lys Val His Lys Thr Tyr Phe Glu Lys 110 115 120 gtt gaa aga tta aaa cac gga acc ttt ggc ccc gtt cat ttt agg aat 438 Val Glu Arg Leu Lys His Gly Thr Phe Gly Pro Val His Phe Arg Asn 125 130 135 caa gtc aag ata aga cgg aga gtt gat gta aac cct ggt cac gcg gac 486 Gln Val Lys Ile Arg Arg Arg Val Asp Val Asn Pro Gly His Ala Asp 140 145 150 ctc agt gcc aaa gaa gca caa gat gtg atc atg gaa gtt gtt ttc cca 534 Leu Ser Ala Lys Glu Ala Gln Asp Val Ile Met Glu Val Val Phe Pro 155 160 165 aat gaa gtg gga gcc aga att cta aca tcg gaa tca caa cta aca ata 582 Asn Glu Val Gly Ala Arg Ile Leu Thr Ser Glu Ser Gln Leu Thr Ile 170 175 180 185 acc aaa gag aaa aaa gaa gaa ctt cag gac tgc aaa att gcc ccc ttg 630 Thr Lys Glu Lys Lys Glu Glu Leu Gln Asp Cys Lys Ile Ala Pro Leu 190 195 200 atg gta gca tac atg cta gaa aga gag ttg gtc cga aaa aca aga ttc 678 Met Val Ala Tyr Met Leu Glu Arg Glu Leu Val Arg Lys Thr Arg Phe 205 210 215 ctc cca gtg gct ggc gga aca agc agt gta tac att gaa gtg ttg cat 726 Leu Pro Val Ala Gly Gly Thr Ser Ser Val Tyr Ile Glu Val Leu His 220 225 230 ctg act cag gga aca tgc tgg gaa caa atg tac acc cca gga gga gaa 774 Leu Thr Gln Gly Thr Cys Trp Glu Gln Met Tyr Thr Pro Gly Gly Glu 235 240 245 gtt aga aac gat gac att gat caa agt tta att att gct gcc cgg aac 822 Val Arg Asn Asp Asp Ile Asp Gln Ser Leu Ile Ile Ala Ala Arg Asn 250 255 260 265 ata gtg aga aga gcg aca gta tca gca gat cca cta gca tcc ctg ctg 870 Ile Val Arg Arg Ala Thr Val Ser Ala Asp Pro Leu Ala Ser Leu Leu 270 275 280 gaa atg tgc cac agt aca cag att ggt gga ata agg atg gta gac atc 918 Glu Met Cys His Ser Thr Gln Ile Gly Gly Ile Arg Met Val Asp Ile 285 290 295 ctt aag cag aat cca aca gag gaa caa gct gtg gat ata tgc aaa gca 966 Leu Lys Gln Asn Pro Thr Glu Glu Gln Ala Val Asp Ile Cys Lys Ala 300 305 310 gca atg ggg tta aga att agc tca tca ttc agc ttt ggt gga ttc acc 1014 Ala Met Gly Leu Arg Ile Ser Ser Ser Phe Ser Phe Gly Gly Phe Thr 315 320 325 ttt aag aga aca agt gga tca tca gtc aag aga gaa gaa gaa atg ctt 1062 Phe Lys Arg Thr Ser Gly Ser Ser Val Lys Arg Glu Glu Glu Met Leu 330 335 340 345 acg ggc aac ctt caa aca ttg aaa ata aga gtg cat gaa ggc tat gaa 1110 Thr Gly Asn Leu Gln Thr Leu Lys Ile Arg Val His Glu Gly Tyr Glu 350 355 360 gaa ttc aca atg gtc gga aga aga gca aca gcc att ctc aga aag gca 1158 Glu Phe Thr Met Val Gly Arg Arg Ala Thr Ala Ile Leu Arg Lys Ala 365 370 375 acc aga aga ttg att caa ttg ata gta agt ggg aga gat gaa caa tca 1206 Thr Arg Arg Leu Ile Gln Leu Ile Val Ser Gly Arg Asp Glu Gln Ser 380 385 390 att gct gaa gca ata att gta gcc atg gtg ttt tc 1241 Ile Ala Glu Ala Ile Ile Val Ala Met Val Phe 395 400 17 404 PRT Equine influenza virus H3N8 17 Met Glu Arg Ile Lys Glu Leu Arg Asp Leu Met Ser Gln Ser Arg Thr 1 5 10 15 Arg Glu Ile Leu Thr Lys Thr Thr Val Asp His Met Ala Ile Ile Lys 20 25 30 Lys Tyr Thr Ser Gly Arg Gln Glu Lys Asn Pro Ala Leu Arg Met Lys 35 40 45 Trp Met Met Ala Met Lys Tyr Pro Ile Thr Ala Asp Lys Arg Ile Met 50 55 60 Glu Met Ile Pro Glu Arg Asn Glu Gln Gly Gln Thr Leu Trp Ser Lys 65 70 75 80 Thr Asn Asp Ala Gly Ser Asp Arg Val Met Val Ser Pro Leu Ala Val 85 90 95 Thr Trp Trp Asn Arg Asn Gly Pro Thr Thr Ser Thr Ile His Tyr Pro 100 105 110 Lys Val His Lys Thr Tyr Phe Glu Lys Val Glu Arg Leu Lys His Gly 115 120 125 Thr Phe Gly Pro Val His Phe Arg Asn Gln Val Lys Ile Arg Arg Arg 130 135 140 Val Asp Val Asn Pro Gly His Ala Asp Leu Ser Ala Lys Glu Ala Gln 145 150 155 160 Asp Val Ile Met Glu Val Val Phe Pro Asn Glu Val Gly Ala Arg Ile 165 170 175 Leu Thr Ser Glu Ser Gln Leu Thr Ile Thr Lys Glu Lys Lys Glu Glu 180 185 190 Leu Gln Asp Cys Lys Ile Ala Pro Leu Met Val Ala Tyr Met Leu Glu 195 200 205 Arg Glu Leu Val Arg Lys Thr Arg Phe Leu Pro Val Ala Gly Gly Thr 210 215 220 Ser Ser Val Tyr Ile Glu Val Leu His Leu Thr Gln Gly Thr Cys Trp 225 230 235 240 Glu Gln Met Tyr Thr Pro Gly Gly Glu Val Arg Asn Asp Asp Ile Asp 245 250 255 Gln Ser Leu Ile Ile Ala Ala Arg Asn Ile Val Arg Arg Ala Thr Val 260 265 270 Ser Ala Asp Pro Leu Ala Ser Leu Leu Glu Met Cys His Ser Thr Gln 275 280 285 Ile Gly Gly Ile Arg Met Val Asp Ile Leu Lys Gln Asn Pro Thr Glu 290 295 300 Glu Gln Ala Val Asp Ile Cys Lys Ala Ala Met Gly Leu Arg Ile Ser 305 310 315 320 Ser Ser Phe Ser Phe Gly Gly Phe Thr Phe Lys Arg Thr Ser Gly Ser 325 330 335 Ser Val Lys Arg Glu Glu Glu Met Leu Thr Gly Asn Leu Gln Thr Leu 340 345 350 Lys Ile Arg Val His Glu Gly Tyr Glu Glu Phe Thr Met Val Gly Arg 355 360 365 Arg Ala Thr Ala Ile Leu Arg Lys Ala Thr Arg Arg Leu Ile Gln Leu 370 375 380 Ile Val Ser Gly Arg Asp Glu Gln Ser Ile Ala Glu Ala Ile Ile Val 385 390 395 400 Ala Met Val Phe 18 1214 DNA Equine influenza virus H3N8 18 atggagagaa taaaagaact gagagatcta atgtcacaat cccgcacccg cgagatacta 60 acaaaaacta ctgtggacca catggccata atcaagaaat acacatcagg aagacaagag 120 aagaaccccg cacttaggat gaagtggatg atggcaatga aatacccaat tacagcagat 180 aagaggataa tggaaatgat tcctgagaga aatgaacagg ggcaaaccct ttggagcaaa 240 acgaacgatg ctggctcaga ccgcgtaatg gtatcacctc tggcagtgac atggtggaat 300 aggaatggac caacaacgag cacaattcat tatccaaaag tccacaaaac ttattttgaa 360 aaagttgaaa gattaaaaca cggaaccttt ggccccgttc attttaggaa tcaagtcaag 420 ataagacgga gagttgatgt aaaccctggt cacgcggacc tcagtgccaa agaagcacaa 480 gatgtgatca tggaagttgt tttcccaaat gaagtgggag ccagaattct aacatcggaa 540 tcacaactaa caataaccaa agagaaaaaa gaagaacttc aggactgcaa aattgccccc 600 ttgatggtag catacatgct agaaagagag ttggtccgaa aaacaagatt cctcccagtg 660 gctggcggaa caagcagtgt atacattgaa gtgttgcatc tgactcaggg aacatgctgg 720 gaacaaatgt acaccccagg aggagaagtt agaaacgatg acattgatca aagtttaatt 780 attgctgccc ggaacatagt gagaagagcg acagtatcag cagatccact agcatccctg 840 ctggaaatgt gccacagtac acagattggt ggaataagga tggtagacat ccttaagcag 900 aatccaacag aggaacaagc tgtggatata tgcaaagcag caatggggtt aagaattagc 960 tcatcattca gctttggtgg attcaccttt aagagaacaa gtggatcatc agtcaagaga 1020 gaagaagaaa tgcttacggg caaccttcaa acattgaaaa taagagtgca tgaaggctat 1080 gaagaattca caatggtcgg aagaagagca acagccattc tcagaaaggc aaccagaaga 1140 ttgattcaat tgatagtaag tgggagagat gaacaatcaa ttgctgaagc aataattgta 1200 gccatggtgt tttc 1214 19 1233 DNA Equine influenza virus H3N8 CDS (3)..(1196) 19 ta gaa ttc aca atg gtc gga aga aga gca aca gcc att ctc aga aag 47 Glu Phe Thr Met Val Gly Arg Arg Ala Thr Ala Ile Leu Arg Lys 1 5 10 15 gca acc aga aga ttg att caa ttg ata gta agt ggg aga gat gaa caa 95 Ala Thr Arg Arg Leu Ile Gln Leu Ile Val Ser Gly Arg Asp Glu Gln 20 25 30 tca att gct gaa gca ata att gta gcc atg gtg ttt tcg caa gaa gat 143 Ser Ile Ala Glu Ala Ile Ile Val Ala Met Val Phe Ser Gln Glu Asp 35 40 45 tgc atg ata aaa gca gtt cga ggc gat ttg aac ttc gtt aat aga gca 191 Cys Met Ile Lys Ala Val Arg Gly Asp Leu Asn Phe Val Asn Arg Ala 50 55 60 aat cag cgc ttg aac ccc atg cat caa ctc ttg agg cat ttc caa aaa 239 Asn Gln Arg Leu Asn Pro Met His Gln Leu Leu Arg His Phe Gln Lys 65 70 75 gat gca aaa gtg ctt ttc cag aat tgg ggg att gaa ccc atc gac aat 287 Asp Ala Lys Val Leu Phe Gln Asn Trp Gly Ile Glu Pro Ile Asp Asn 80 85 90 95 gtg atg gga atg att gga ata ttg cct gac atg acc cca agc acc gag 335 Val Met Gly Met Ile Gly Ile Leu Pro Asp Met Thr Pro Ser Thr Glu 100 105 110 atg tca ttg aga gga gtg aga gtc agc aaa atg gga gtg gat gag tac 383 Met Ser Leu Arg Gly Val Arg Val Ser Lys Met Gly Val Asp Glu Tyr 115 120 125 tcc agc act gag aga gtg gtg gtg agc att gac cgt ttt tta aga gtt 431 Ser Ser Thr Glu Arg Val Val Val Ser Ile Asp Arg Phe Leu Arg Val 130 135 140 cgg gat caa agg gga aac ata cta ctg tcc cct gaa gag gtc agt gaa 479 Arg Asp Gln Arg Gly Asn Ile Leu Leu Ser Pro Glu Glu Val Ser Glu 145 150 155 aca caa gga acg gaa aag ctg aca ata att tat tca tca tca atg atg 527 Thr Gln Gly Thr Glu Lys Leu Thr Ile Ile Tyr Ser Ser Ser Met Met 160 165 170 175 tgg gag att aat ggt ccc gaa tca gtg ttg gtc aat act tat caa tgg 575 Trp Glu Ile Asn Gly Pro Glu Ser Val Leu Val Asn Thr Tyr Gln Trp 180 185 190 atc atc agg aac tgg gaa att gtg aaa att caa tgg tca cag gat ccc 623 Ile Ile Arg Asn Trp Glu Ile Val Lys Ile Gln Trp Ser Gln Asp Pro 195 200 205 aca atg tta tac aat aag ata gaa ttt gag cca ttc cag tcc ctg gtc 671 Thr Met Leu Tyr Asn Lys Ile Glu Phe Glu Pro Phe Gln Ser Leu Val 210 215 220 cct agg gcc acc aga agc caa tac agc ggt ttc gta aga acc ctg ttt 719 Pro Arg Ala Thr Arg Ser Gln Tyr Ser Gly Phe Val Arg Thr Leu Phe 225 230 235 cag caa atg cga gat gta ctt gga aca ttt gat act gct caa ata ata 767 Gln Gln Met Arg Asp Val Leu Gly Thr Phe Asp Thr Ala Gln Ile Ile 240 245 250 255 aaa ctc ctc cct ttt gcc gct gct cct ccg gaa cag agt agg atg cag 815 Lys Leu Leu Pro Phe Ala Ala Ala Pro Pro Glu Gln Ser Arg Met Gln 260 265 270 ttc tct tct ttg act gtt aat gta aga gga tcg gga atg agg ata ctt 863 Phe Ser Ser Leu Thr Val Asn Val Arg Gly Ser Gly Met Arg Ile Leu 275 280 285 gta aga ggc aat tcc cca gtg ttc aac tac aat aaa gcc act aag agg 911 Val Arg Gly Asn Ser Pro Val Phe Asn Tyr Asn Lys Ala Thr Lys Arg 290 295 300 ctc aca gtc ctc gga aag gat gca ggt gcg ctt act gaa gac cca gat 959 Leu Thr Val Leu Gly Lys Asp Ala Gly Ala Leu Thr Glu Asp Pro Asp 305 310 315 gaa ggt acg gct gga gta gaa tct gct gtt cta aga ggg ttt ctc att 1007 Glu Gly Thr Ala Gly Val Glu Ser Ala Val Leu Arg Gly Phe Leu Ile 320 325 330 335 tta ggt aaa gaa aac aag aga tat ggc cca gca cta agc atc aat gaa 1055 Leu Gly Lys Glu Asn Lys Arg Tyr Gly Pro Ala Leu Ser Ile Asn Glu 340 345 350 ctg agc aaa ctt gca aaa ggg gag aaa gct aat gtg cta att ggg caa 1103 Leu Ser Lys Leu Ala Lys Gly Glu Lys Ala Asn Val Leu Ile Gly Gln 355 360 365 ggg gac gtg gtg ttg gta atg aaa cgg aaa cgt gac tct agc ata ctt 1151 Gly Asp Val Val Leu Val Met Lys Arg Lys Arg Asp Ser Ser Ile Leu 370 375 380 act gac agc cag aca gcg acc aaa agg att cgg atg gcc atc aat 1196 Thr Asp Ser Gln Thr Ala Thr Lys Arg Ile Arg Met Ala Ile Asn 385 390 395 tagtgttgaa ttgtttaaaa acgaccttgt ttctact 1233 20 398 PRT Equine influenza virus H3N8 20 Glu Phe Thr Met Val Gly Arg Arg Ala Thr Ala Ile Leu Arg Lys Ala 1 5 10 15 Thr Arg Arg Leu Ile Gln Leu Ile Val Ser Gly Arg Asp Glu Gln Ser 20 25 30 Ile Ala Glu Ala Ile Ile Val Ala Met Val Phe Ser Gln Glu Asp Cys 35 40 45 Met Ile Lys Ala Val Arg Gly Asp Leu Asn Phe Val Asn Arg Ala Asn 50 55 60 Gln Arg Leu Asn Pro Met His Gln Leu Leu Arg His Phe Gln Lys Asp 65 70 75 80 Ala Lys Val Leu Phe Gln Asn Trp Gly Ile Glu Pro Ile Asp Asn Val 85 90 95 Met Gly Met Ile Gly Ile Leu Pro Asp Met Thr Pro Ser Thr Glu Met 100 105 110 Ser Leu Arg Gly Val Arg Val Ser Lys Met Gly Val Asp Glu Tyr Ser 115 120 125 Ser Thr Glu Arg Val Val Val Ser Ile Asp Arg Phe Leu Arg Val Arg 130 135 140 Asp Gln Arg Gly Asn Ile Leu Leu Ser Pro Glu Glu Val Ser Glu Thr 145 150 155 160 Gln Gly Thr Glu Lys Leu Thr Ile Ile Tyr Ser Ser Ser Met Met Trp 165 170 175 Glu Ile Asn Gly Pro Glu Ser Val Leu Val Asn Thr Tyr Gln Trp Ile 180 185 190 Ile Arg Asn Trp Glu Ile Val Lys Ile Gln Trp Ser Gln Asp Pro Thr 195 200 205 Met Leu Tyr Asn Lys Ile Glu Phe Glu Pro Phe Gln Ser Leu Val Pro 210 215 220 Arg Ala Thr Arg Ser Gln Tyr Ser Gly Phe Val Arg Thr Leu Phe Gln 225 230 235 240 Gln Met Arg Asp Val Leu Gly Thr Phe Asp Thr Ala Gln Ile Ile Lys 245 250 255 Leu Leu Pro Phe Ala Ala Ala Pro Pro Glu Gln Ser Arg Met Gln Phe 260 265 270 Ser Ser Leu Thr Val Asn Val Arg Gly Ser Gly Met Arg Ile Leu Val 275 280 285 Arg Gly Asn Ser Pro Val Phe Asn Tyr Asn Lys Ala Thr Lys Arg Leu 290 295 300 Thr Val Leu Gly Lys Asp Ala Gly Ala Leu Thr Glu Asp Pro Asp Glu 305 310 315 320 Gly Thr Ala Gly Val Glu Ser Ala Val Leu Arg Gly Phe Leu Ile Leu 325 330 335 Gly Lys Glu Asn Lys Arg Tyr Gly Pro Ala Leu Ser Ile Asn Glu Leu 340 345 350 Ser Lys Leu Ala Lys Gly Glu Lys Ala Asn Val Leu Ile Gly Gln Gly 355 360 365 Asp Val Val Leu Val Met Lys Arg Lys Arg Asp Ser Ser Ile Leu Thr 370 375 380 Asp Ser Gln Thr Ala Thr Lys Arg Ile Arg Met Ala Ile Asn 385 390 395 21 1194 DNA Equine influenza virus H3N8 21 gaattcacaa tggtcggaag aagagcaaca gccattctca gaaaggcaac cagaagattg 60 attcaattga tagtaagtgg gagagatgaa caatcaattg ctgaagcaat aattgtagcc 120 atggtgtttt cgcaagaaga ttgcatgata aaagcagttc gaggcgattt gaacttcgtt 180 aatagagcaa atcagcgctt gaaccccatg catcaactct tgaggcattt ccaaaaagat 240 gcaaaagtgc ttttccagaa ttgggggatt gaacccatcg acaatgtgat gggaatgatt 300 ggaatattgc ctgacatgac cccaagcacc gagatgtcat tgagaggagt gagagtcagc 360 aaaatgggag tggatgagta ctccagcact gagagagtgg tggtgagcat tgaccgtttt 420 ttaagagttc gggatcaaag gggaaacata ctactgtccc ctgaagaggt cagtgaaaca 480 caaggaacgg aaaagctgac aataatttat tcatcatcaa tgatgtggga gattaatggt 540 cccgaatcag tgttggtcaa tacttatcaa tggatcatca ggaactggga aattgtgaaa 600 attcaatggt cacaggatcc cacaatgtta tacaataaga tagaatttga gccattccag 660 tccctggtcc ctagggccac cagaagccaa tacagcggtt tcgtaagaac cctgtttcag 720 caaatgcgag atgtacttgg aacatttgat actgctcaaa taataaaact cctccctttt 780 gccgctgctc ctccggaaca gagtaggatg cagttctctt ctttgactgt taatgtaaga 840 ggatcgggaa tgaggatact tgtaagaggc aattccccag tgttcaacta caataaagcc 900 actaagaggc tcacagtcct cggaaaggat gcaggtgcgc ttactgaaga cccagatgaa 960 ggtacggctg gagtagaatc tgctgttcta agagggtttc tcattttagg taaagaaaac 1020 aagagatatg gcccagcact aagcatcaat gaactgagca aacttgcaaa aggggagaaa 1080 gctaatgtgc taattgggca aggggacgtg gtgttggtaa tgaaacggaa acgtgactct 1140 agcatactta ctgacagcca gacagcgacc aaaaggattc ggatggccat caat 1194 22 1232 DNA Equine influenza virus H3N8 22 agaattcaca atggtcggaa gaagagcaac agccattctc agaaaggcaa ccagaagatt 60 gattcaattg atagtaagtg ggagagatga acaatcaatt gctgaagcaa taattgtagc 120 catggtgttt tcgcaagaag attgcatgat aaaagcagtt cgaggcgatt tgaacttcgt 180 taatagagca aatcagcgct tgaaccccat gcatcaactc ttgaggcatt tccaaaaaga 240 tgcaaaagtg cttttccaga attgggggat tgaacccatc gacaatgtga tgggaatgat 300 tggaatattg cctgacatga ccccaagcac cgagatgtca ttgagaggag tgagagtcag 360 caaaatggga gtggatgagt actccagcac tgagagagtg gtggtgagca ttgaccgttt 420 tttaagagtt cgggatcaaa ggggaaacat actactgtcc cctgaagagg tcagtgaaac 480 acaaggaacg gaaaagctga caataattta ttcatcatca atgatgtggg agattaatgg 540 tcccgaatca gtgttggtca atacttatca atggatcatc aggaactggg aaattgtgaa 600 aattcaatgg tcacaggatc ccacaatgtt atacaataag atagaatttg agccattcca 660 gtccctggtc cctagggcca ccagaagcca atacagcggt ttcgtaagaa ccctgtttca 720 gcaaatgcga gatgtacttg gaacatttga tactgctcaa ataataaaac tcctcccttt 780 tgccgctgct cctccggaac agagtaggat gcagttctct tctttgactg ttaatgtaag 840 aggatcggga atgaggatac ttgtaagagg caattcccca gtgttcaact acaataaagc 900 cactaagagg ctcacagtcc tcggaaagga tgcaggtgcg cttactgaag acccagatga 960 aggtacggct ggagtagaat ctgctgttct aagagggttt ctcattttag gtaaagaaaa 1020 caagagatat ggcccagcac taagcatcaa tgaactgagc aaacttgcaa aaggggagaa 1080 agctaatgtg ctaattgggc aaggggacgt ggtgttggta atgaaacgga aacgtgactc 1140 tagcatactt actgacagcc agacagcgac caaaaggatt cggatggcca tcaattagtg 1200 ttgaattgtt taaaaacgac cttgtttcta ct 1232 23 1232 DNA Equine influenza virus H3N8 CDS (2)..(1195) 23 a gaa ttc aca atg gtc gga aga aga gca aca gcc att ctc aga aag gca 49 Glu Phe Thr Met Val Gly Arg Arg Ala Thr Ala Ile Leu Arg Lys Ala 1 5 10 15 acc aga aga ttg att caa ttg ata gta agt ggg aga gat gaa caa tca 97 Thr Arg Arg Leu Ile Gln Leu Ile Val Ser Gly Arg Asp Glu Gln Ser 20 25 30 att gct gaa gca ata att gta gcc atg gtg ttt tcg caa gaa gat tgc 145 Ile Ala Glu Ala Ile Ile Val Ala Met Val Phe Ser Gln Glu Asp Cys 35 40 45 atg ata caa gca gtt cga ggc gat ttg aac ttc gtt aat aga gca aat 193 Met Ile Gln Ala Val Arg Gly Asp Leu Asn Phe Val Asn Arg Ala Asn 50 55 60 cag cgc ttg aac ccc atg cat caa ctc ttg agg cat ttc caa aaa gat 241 Gln Arg Leu Asn Pro Met His Gln Leu Leu Arg His Phe Gln Lys Asp 65 70 75 80 gca aaa gtg ctt ttc cag aat tgg ggg att gaa ccc atc gac aat gtg 289 Ala Lys Val Leu Phe Gln Asn Trp Gly Ile Glu Pro Ile Asp Asn Val 85 90 95 atg gga atg att gga ata ttg cct gac atg acc cca agc acc gag atg 337 Met Gly Met Ile Gly Ile Leu Pro Asp Met Thr Pro Ser Thr Glu Met 100 105 110 tca ttg aga gga gtg aga gtc agc aaa atg gga gtg gat gag tac tcc 385 Ser Leu Arg Gly Val Arg Val Ser Lys Met Gly Val Asp Glu Tyr Ser 115 120 125 agc act gag aga gtg gtg gtg agc att gac cgt ttt tta aga gtt cgg 433 Ser Thr Glu Arg Val Val Val Ser Ile Asp Arg Phe Leu Arg Val Arg 130 135 140 gat caa agg gga aac ata cta ctg tcc cct gaa gag gtc agt gaa aca 481 Asp Gln Arg Gly Asn Ile Leu Leu Ser Pro Glu Glu Val Ser Glu Thr 145 150 155 160 caa gga acg gaa aag ctg aca ata att tat tca tca tca atg atg tgg 529 Gln Gly Thr Glu Lys Leu Thr Ile Ile Tyr Ser Ser Ser Met Met Trp 165 170 175 gag att aat ggt ccc gaa tca gtg ttg gtc aat act tat caa tgg atc 577 Glu Ile Asn Gly Pro Glu Ser Val Leu Val Asn Thr Tyr Gln Trp Ile 180 185 190 atc agg aac tgg gaa att gtg aaa att caa tgg tca cag gat ccc aca 625 Ile Arg Asn Trp Glu Ile Val Lys Ile Gln Trp Ser Gln Asp Pro Thr 195 200 205 atg tta tac aat aag ata gaa ttt gag cca ttc cag tcc ctg gtc cct 673 Met Leu Tyr Asn Lys Ile Glu Phe Glu Pro Phe Gln Ser Leu Val Pro 210 215 220 agg gcc acc aga agc caa tac agc ggt ttc gta aga acc ctg ttt cag 721 Arg Ala Thr Arg Ser Gln Tyr Ser Gly Phe Val Arg Thr Leu Phe Gln 225 230 235 240 caa atg cga gat gta ctt gga aca ttt gat act gct caa ata ata aaa 769 Gln Met Arg Asp Val Leu Gly Thr Phe Asp Thr Ala Gln Ile Ile Lys 245 250 255 ctc ctc cct ttt gcc gct gct cct ccg gaa cag agt agg atg cag ttc 817 Leu Leu Pro Phe Ala Ala Ala Pro Pro Glu Gln Ser Arg Met Gln Phe 260 265 270 tct tct ttg act gtt aat gta aga gga tcg gga atg agg ata ctt gta 865 Ser Ser Leu Thr Val Asn Val Arg Gly Ser Gly Met Arg Ile Leu Val 275 280 285 aga ggc aat tcc cca gtg ttc aac tac aat aaa gcc act aag agg ctc 913 Arg Gly Asn Ser Pro Val Phe Asn Tyr Asn Lys Ala Thr Lys Arg Leu 290 295 300 aca gtc ctc gga aaa gat gca ggt gcg ctt act gaa gac cca gat gaa 961 Thr Val Leu Gly Lys Asp Ala Gly Ala Leu Thr Glu Asp Pro Asp Glu 305 310 315 320 ggt acg gct gga gta gaa tct gct gtt cta aga ggg ttt ctc att tta 1009 Gly Thr Ala Gly Val Glu Ser Ala Val Leu Arg Gly Phe Leu Ile Leu 325 330 335 ggt aaa gaa aac aag aga tat ggc cca gca cta agc atc aat gaa ctg 1057 Gly Lys Glu Asn Lys Arg Tyr Gly Pro Ala Leu Ser Ile Asn Glu Leu 340 345 350 agc aaa ctt gca aaa ggg gag aaa gct aat gtg cta att ggg caa ggg 1105 Ser Lys Leu Ala Lys Gly Glu Lys Ala Asn Val Leu Ile Gly Gln Gly 355 360 365 gac gtg gtg ttg gta atg aaa cgg aaa cgt gac tct agc ata ctt act 1153 Asp Val Val Leu Val Met Lys Arg Lys Arg Asp Ser Ser Ile Leu Thr 370 375 380 gac agc cag aca gcg acc aaa agg att cgg atg gcc atc aat 1195 Asp Ser Gln Thr Ala Thr Lys Arg Ile Arg Met Ala Ile Asn 385 390 395 tagtgttgaa ttgtttaaaa acgaccttgt ttctact 1232 24 398 PRT Equine influenza virus H3N8 24 Glu Phe Thr Met Val Gly Arg Arg Ala Thr Ala Ile Leu Arg Lys Ala 1 5 10 15 Thr Arg Arg Leu Ile Gln Leu Ile Val Ser Gly Arg Asp Glu Gln Ser 20 25 30 Ile Ala Glu Ala Ile Ile Val Ala Met Val Phe Ser Gln Glu Asp Cys 35 40 45 Met Ile Gln Ala Val Arg Gly Asp Leu Asn Phe Val Asn Arg Ala Asn 50 55 60 Gln Arg Leu Asn Pro Met His Gln Leu Leu Arg His Phe Gln Lys Asp 65 70 75 80 Ala Lys Val Leu Phe Gln Asn Trp Gly Ile Glu Pro Ile Asp Asn Val 85 90 95 Met Gly Met Ile Gly Ile Leu Pro Asp Met Thr Pro Ser Thr Glu Met 100 105 110 Ser Leu Arg Gly Val Arg Val Ser Lys Met Gly Val Asp Glu Tyr Ser 115 120 125 Ser Thr Glu Arg Val Val Val Ser Ile Asp Arg Phe Leu Arg Val Arg 130 135 140 Asp Gln Arg Gly Asn Ile Leu Leu Ser Pro Glu Glu Val Ser Glu Thr 145 150 155 160 Gln Gly Thr Glu Lys Leu Thr Ile Ile Tyr Ser Ser Ser Met Met Trp 165 170 175 Glu Ile Asn Gly Pro Glu Ser Val Leu Val Asn Thr Tyr Gln Trp Ile 180 185 190 Ile Arg Asn Trp Glu Ile Val Lys Ile Gln Trp Ser Gln Asp Pro Thr 195 200 205 Met Leu Tyr Asn Lys Ile Glu Phe Glu Pro Phe Gln Ser Leu Val Pro 210 215 220 Arg Ala Thr Arg Ser Gln Tyr Ser Gly Phe Val Arg Thr Leu Phe Gln 225 230 235 240 Gln Met Arg Asp Val Leu Gly Thr Phe Asp Thr Ala Gln Ile Ile Lys 245 250 255 Leu Leu Pro Phe Ala Ala Ala Pro Pro Glu Gln Ser Arg Met Gln Phe 260 265 270 Ser Ser Leu Thr Val Asn Val Arg Gly Ser Gly Met Arg Ile Leu Val 275 280 285 Arg Gly Asn Ser Pro Val Phe Asn Tyr Asn Lys Ala Thr Lys Arg Leu 290 295 300 Thr Val Leu Gly Lys Asp Ala Gly Ala Leu Thr Glu Asp Pro Asp Glu 305 310 315 320 Gly Thr Ala Gly Val Glu Ser Ala Val Leu Arg Gly Phe Leu Ile Leu 325 330 335 Gly Lys Glu Asn Lys Arg Tyr Gly Pro Ala Leu Ser Ile Asn Glu Leu 340 345 350 Ser Lys Leu Ala Lys Gly Glu Lys Ala Asn Val Leu Ile Gly Gln Gly 355 360 365 Asp Val Val Leu Val Met Lys Arg Lys Arg Asp Ser Ser Ile Leu Thr 370 375 380 Asp Ser Gln Thr Ala Thr Lys Arg Ile Arg Met Ala Ile Asn 385 390 395 25 1194 DNA Equine influenza virus H3N8 25 gaattcacaa tggtcggaag aagagcaaca gccattctca gaaaggcaac cagaagattg 60 attcaattga tagtaagtgg gagagatgaa caatcaattg ctgaagcaat aattgtagcc 120 atggtgtttt cgcaagaaga ttgcatgata caagcagttc gaggcgattt gaacttcgtt 180 aatagagcaa atcagcgctt gaaccccatg catcaactct tgaggcattt ccaaaaagat 240 gcaaaagtgc ttttccagaa ttgggggatt gaacccatcg acaatgtgat gggaatgatt 300 ggaatattgc ctgacatgac cccaagcacc gagatgtcat tgagaggagt gagagtcagc 360 aaaatgggag tggatgagta ctccagcact gagagagtgg tggtgagcat tgaccgtttt 420 ttaagagttc gggatcaaag gggaaacata ctactgtccc ctgaagaggt cagtgaaaca 480 caaggaacgg aaaagctgac aataatttat tcatcatcaa tgatgtggga gattaatggt 540 cccgaatcag tgttggtcaa tacttatcaa tggatcatca ggaactggga aattgtgaaa 600 attcaatggt cacaggatcc cacaatgtta tacaataaga tagaatttga gccattccag 660 tccctggtcc ctagggccac cagaagccaa tacagcggtt tcgtaagaac cctgtttcag 720 caaatgcgag atgtacttgg aacatttgat actgctcaaa taataaaact cctccctttt 780 gccgctgctc ctccggaaca gagtaggatg cagttctctt ctttgactgt taatgtaaga 840 ggatcgggaa tgaggatact tgtaagaggc aattccccag tgttcaacta caataaagcc 900 actaagaggc tcacagtcct cggaaaagat gcaggtgcgc ttactgaaga cccagatgaa 960 ggtacggctg gagtagaatc tgctgttcta agagggtttc tcattttagg taaagaaaac 1020 aagagatatg gcccagcact aagcatcaat gaactgagca aacttgcaaa aggggagaaa 1080 gctaatgtgc taattgggca aggggacgtg gtgttggtaa tgaaacggaa acgtgactct 1140 agcatactta ctgacagcca gacagcgacc aaaaggattc ggatggccat caat 1194 26 23 DNA Artificial sequence Synthetic Primer 26 agcaaaagca ggtagatatt gaa 23 27 24 DNA Artificial sequence Synthetic Primer 27 agtagaaaca aggtagtttt ttac 24 28 18 DNA Artificial sequence Synthetic Primer 28 caggaaacag ctatgacc 18 29 20 DNA Artificial sequence Synthetic Primer 29 taatacgact cactataggg 20 30 18 DNA Artificial sequence Synthetic Primer 30 tggtgcacta gccagctg 18 31 18 DNA Artificial sequence Synthetic Primer 31 ttgcctgtac catctgcc 18 32 23 DNA Artificial sequence Synthetic Primer 32 agcaaaagca ggggatattt ctg 23 33 23 DNA Artificial sequence Synthetic Primer 33 agtagaaaca agggtgtttt taa 23 34 16 DNA Artificial sequence Synthetic Primer 34 gacatccctg actatg 16 35 16 DNA Artificial sequence Synthetic Primer 35 gcatctgtta agtcaa 16 36 25 DNA Artificial sequence Synthetic Primer 36 agcaaaagca ggtcaaatat attca 25 37 26 DNA Artificial sequence Synthetic Primer 37 gaaaacacca tggctacaat tattgc 26 38 27 DNA Artificial sequence Synthetic Primer 38 agaattcaca atggtcggaa gaagagc 27 39 27 DNA Artificial sequence Synthetic Primer 39 agtagaaaca aggtcgtttt taaacaa 27 40 19 DNA Artificial sequence Synthetic Primer 40 agccgtacct tcatctggg 19 41 19 DNA Artificial sequence Synthetic Primer 41 agcactgaga gagtggtgg 19 42 19 DNA Artificial sequence Synthetic Primer 42 gtaagaggca attccccag 19 43 18 DNA Artificial sequence Synthetic Primer 43 cagcttttcc gttccttg 18 44 2341 DNA Equine influenza virus H3N8 CDS (28)..(2304) 44 agcaaaagca ggtcaaatat attcaat atg gag aga ata aaa gaa ctg aga gat 54 Met Glu Arg Ile Lys Glu Leu Arg Asp 1 5 cta atg tca caa tcc cgc acc cgc gag ata cta aca aaa act act gtg 102 Leu Met Ser Gln Ser Arg Thr Arg Glu Ile Leu Thr Lys Thr Thr Val 10 15 20 25 gac cac atg gcc ata atc aag aaa tac aca tca gga aga caa gag aag 150 Asp His Met Ala Ile Ile Lys Lys Tyr Thr Ser Gly Arg Gln Glu Lys 30 35 40 aac ccc gca ctt agg atg aag tgg atg atg gca atg aaa tac cca att 198 Asn Pro Ala Leu Arg Met Lys Trp Met Met Ala Met Lys Tyr Pro Ile 45 50 55 aca gca gat aag agg ata atg gaa atg att cct gag aga aat gaa cag 246 Thr Ala Asp Lys Arg Ile Met Glu Met Ile Pro Glu Arg Asn Glu Gln 60 65 70 ggg caa acc ctt tgg agc aaa acg aac gat gct ggc tca gac cgc gta 294 Gly Gln Thr Leu Trp Ser Lys Thr Asn Asp Ala Gly Ser Asp Arg Val 75 80 85 atg gta tca cct ctg gca gtg aca tgg tgg aat agg aat gga cca aca 342 Met Val Ser Pro Leu Ala Val Thr Trp Trp Asn Arg Asn Gly Pro Thr 90 95 100 105 acg agc aca att cat tat cca aaa gtc tac aaa act tat ttt gaa aaa 390 Thr Ser Thr Ile His Tyr Pro Lys Val Tyr Lys Thr Tyr Phe Glu Lys 110 115 120 gtt gaa aga tta aaa cac gga acc ttt ggc ccc gtt cat ttt agg aat 438 Val Glu Arg Leu Lys His Gly Thr Phe Gly Pro Val His Phe Arg Asn 125 130 135 caa gtc aag ata aga cgg aga gtt gat gta aac cct ggt cac gcg gac 486 Gln Val Lys Ile Arg Arg Arg Val Asp Val Asn Pro Gly His Ala Asp 140 145 150 ctc agt gcc aaa gaa gca caa gat gtg atc atg gaa gtt gtt ttc cca 534 Leu Ser Ala Lys Glu Ala Gln Asp Val Ile Met Glu Val Val Phe Pro 155 160 165 aat gaa gtg gga gcc aga att cta aca tcg gaa tca caa cta aca ata 582 Asn Glu Val Gly Ala Arg Ile Leu Thr Ser Glu Ser Gln Leu Thr Ile 170 175 180 185 acc aaa gag aaa aaa gaa gaa ctt cag gac tgc aaa att gcc ccc ttg 630 Thr Lys Glu Lys Lys Glu Glu Leu Gln Asp Cys Lys Ile Ala Pro Leu 190 195 200 atg gta gca tac atg cta gaa aga gag ttg gtc cga aaa aca aga ttc 678 Met Val Ala Tyr Met Leu Glu Arg Glu Leu Val Arg Lys Thr Arg Phe 205 210 215 ctc cca gtg gct ggc gga aca agc agt gta tac att gaa gtg ttg cat 726 Leu Pro Val Ala Gly Gly Thr Ser Ser Val Tyr Ile Glu Val Leu His 220 225 230 ctg act cag gga aca tgc tgg gaa caa atg tac acc cca gga gga gaa 774 Leu Thr Gln Gly Thr Cys Trp Glu Gln Met Tyr Thr Pro Gly Gly Glu 235 240 245 gtt aga aac gat gac att gat caa agt tta att att gct gcc cgg aac 822 Val Arg Asn Asp Asp Ile Asp Gln Ser Leu Ile Ile Ala Ala Arg Asn 250 255 260 265 ata gtg aga aga gcg aca gta tca gca gat cca cta gca tcc ctg ctg 870 Ile Val Arg Arg Ala Thr Val Ser Ala Asp Pro Leu Ala Ser Leu Leu 270 275 280 gaa atg tgc cac agt aca cag att ggt gga ata agg atg gta gac atc 918 Glu Met Cys His Ser Thr Gln Ile Gly Gly Ile Arg Met Val Asp Ile 285 290 295 ctt aag cag aat cca aca gag gaa caa gct gtg gat ata tgc aaa gca 966 Leu Lys Gln Asn Pro Thr Glu Glu Gln Ala Val Asp Ile Cys Lys Ala 300 305 310 gca atg ggg tta aga att agc tca tca ttc agc ttt ggt gga ttc acc 1014 Ala Met Gly Leu Arg Ile Ser Ser Ser Phe Ser Phe Gly Gly Phe Thr 315 320 325 ttt aag aga aca agt gga tca tca gtc aag aga gaa gaa gaa atg ctt 1062 Phe Lys Arg Thr Ser Gly Ser Ser Val Lys Arg Glu Glu Glu Met Leu 330 335 340 345 acg ggc aac ctt caa aca ttg aaa ata aga gtg cat gaa ggc tat gaa 1110 Thr Gly Asn Leu Gln Thr Leu Lys Ile Arg Val His Glu Gly Tyr Glu 350 355 360 gaa ttc aca atg gtc gga aga aga gca aca gcc att ctc aga aag gca 1158 Glu Phe Thr Met Val Gly Arg Arg Ala Thr Ala Ile Leu Arg Lys Ala 365 370 375 acc aga aga ttg att caa ttg ata gta agt ggg aga gat gaa caa tca 1206 Thr Arg Arg Leu Ile Gln Leu Ile Val Ser Gly Arg Asp Glu Gln Ser 380 385 390 att gct gaa gca ata att gta gcc atg gtg ttt tcg caa gaa gat tgc 1254 Ile Ala Glu Ala Ile Ile Val Ala Met Val Phe Ser Gln Glu Asp Cys 395 400 405 atg ata aaa gca gtt cga ggc gat ttg aac ttc gtt aat aga gca aat 1302 Met Ile Lys Ala Val Arg Gly Asp Leu Asn Phe Val Asn Arg Ala Asn 410 415 420 425 cag cgc ttg aac ccc atg cat caa ctc ttg agg cat ttc caa aaa gat 1350 Gln Arg Leu Asn Pro Met His Gln Leu Leu Arg His Phe Gln Lys Asp 430 435 440 gca aaa gtg ctt ttc cag aat tgg ggg att gaa ccc atc gac aat gtg 1398 Ala Lys Val Leu Phe Gln Asn Trp Gly Ile Glu Pro Ile Asp Asn Val 445 450 455 atg gga atg att gga ata ttg cct gac atg acc cca agc acc gag atg 1446 Met Gly Met Ile Gly Ile Leu Pro Asp Met Thr Pro Ser Thr Glu Met 460 465 470 tca ttg aga gga gtg aga gtc agc aaa atg gga gtg gat gag tac tcc 1494 Ser Leu Arg Gly Val Arg Val Ser Lys Met Gly Val Asp Glu Tyr Ser 475 480 485 agc act gag aga gtg gtg gtg agc att gac cgt ttt tta aga gtt cgg 1542 Ser Thr Glu Arg Val Val Val Ser Ile Asp Arg Phe Leu Arg Val Arg 490 495 500 505 gat caa agg gga aac ata cta ctg tcc cct gaa gag gtc agt gaa aca 1590 Asp Gln Arg Gly Asn Ile Leu Leu Ser Pro Glu Glu Val Ser Glu Thr 510 515 520 caa gga acg gaa aag ctg aca ata att tat tca tca tca atg atg tgg 1638 Gln Gly Thr Glu Lys Leu Thr Ile Ile Tyr Ser Ser Ser Met Met Trp 525 530 535 gag att aat ggt ccc gaa tca gtg ttg gtc aat act tat caa tgg atc 1686 Glu Ile Asn Gly Pro Glu Ser Val Leu Val Asn Thr Tyr Gln Trp Ile 540 545 550 atc agg aac tgg gaa att gtg aaa att caa tgg tca cag gat ccc aca 1734 Ile Arg Asn Trp Glu Ile Val Lys Ile Gln Trp Ser Gln Asp Pro Thr 555 560 565 atg tta tac aat aag ata gaa ttt gag cca ttc cag tcc ctg gtc cct 1782 Met Leu Tyr Asn Lys Ile Glu Phe Glu Pro Phe Gln Ser Leu Val Pro 570 575 580 585 agg gcc acc aga agc caa tac agc ggt ttc gta aga acc ctg ttt cag 1830 Arg Ala Thr Arg Ser Gln Tyr Ser Gly Phe Val Arg Thr Leu Phe Gln 590 595 600 caa atg cga gat gta ctt gga aca ttt gat act gct caa ata ata aaa 1878 Gln Met Arg Asp Val Leu Gly Thr Phe Asp Thr Ala Gln Ile Ile Lys 605 610 615 ctc ctc cct ttt gcc gct gct cct ccg gaa cag agt agg atg cag ttc 1926 Leu Leu Pro Phe Ala Ala Ala Pro Pro Glu Gln Ser Arg Met Gln Phe 620 625 630 tct tct ttg act gtt aat gta aga gga tcg gga atg agg ata ctt gta 1974 Ser Ser Leu Thr Val Asn Val Arg Gly Ser Gly Met Arg Ile Leu Val 635 640 645 aga ggc aat tcc cca gtg ttc aac tac aat aaa gcc act aag agg ctc 2022 Arg Gly Asn Ser Pro Val Phe Asn Tyr Asn Lys Ala Thr Lys Arg Leu 650 655 660 665 aca gtc ctc gga aag gat gca ggt gcg ctt act gaa gac cca gat gaa 2070 Thr Val Leu Gly Lys Asp Ala Gly Ala Leu Thr Glu Asp Pro Asp Glu 670 675 680 ggt acg gct gga gta gaa tct gct gtt cta aga ggg ttt ctc att tta 2118 Gly Thr Ala Gly Val Glu Ser Ala Val Leu Arg Gly Phe Leu Ile Leu 685 690 695 ggt aaa gaa aac aag aga tat ggc cca gca cta agc atc aat gaa ctg 2166 Gly Lys Glu Asn Lys Arg Tyr Gly Pro Ala Leu Ser Ile Asn Glu Leu 700 705 710 agc aaa ctt gca aaa ggg gag aaa gct aat gtg cta att ggg caa ggg 2214 Ser Lys Leu Ala Lys Gly Glu Lys Ala Asn Val Leu Ile Gly Gln Gly 715 720 725 gac gtg gtg ttg gta atg aaa cgg aaa cgt gac tct agc ata ctt act 2262 Asp Val Val Leu Val Met Lys Arg Lys Arg Asp Ser Ser Ile Leu Thr 730 735 740 745 gac agc cag aca gcg acc aaa agg att cgg atg gcc atc aat 2304 Asp Ser Gln Thr Ala Thr Lys Arg Ile Arg Met Ala Ile Asn 750 755 tagtgttgaa ttgtttaaaa acgaccttgt ttctact 2341 45 759 PRT Equine influenza virus H3N8 45 Met Glu Arg Ile Lys Glu Leu Arg Asp Leu Met Ser Gln Ser Arg Thr 1 5 10 15 Arg Glu Ile Leu Thr Lys Thr Thr Val Asp His Met Ala Ile Ile Lys 20 25 30 Lys Tyr Thr Ser Gly Arg Gln Glu Lys Asn Pro Ala Leu Arg Met Lys 35 40 45 Trp Met Met Ala Met Lys Tyr Pro Ile Thr Ala Asp Lys Arg Ile Met 50 55 60 Glu Met Ile Pro Glu Arg Asn Glu Gln Gly Gln Thr Leu Trp Ser Lys 65 70 75 80 Thr Asn Asp Ala Gly Ser Asp Arg Val Met Val Ser Pro Leu Ala Val 85 90 95 Thr Trp Trp Asn Arg Asn Gly Pro Thr Thr Ser Thr Ile His Tyr Pro 100 105 110 Lys Val Tyr Lys Thr Tyr Phe Glu Lys Val Glu Arg Leu Lys His Gly 115 120 125 Thr Phe Gly Pro Val His Phe Arg Asn Gln Val Lys Ile Arg Arg Arg 130 135 140 Val Asp Val Asn Pro Gly His Ala Asp Leu Ser Ala Lys Glu Ala Gln 145 150 155 160 Asp Val Ile Met Glu Val Val Phe Pro Asn Glu Val Gly Ala Arg Ile 165 170 175 Leu Thr Ser Glu Ser Gln Leu Thr Ile Thr Lys Glu Lys Lys Glu Glu 180 185 190 Leu Gln Asp Cys Lys Ile Ala Pro Leu Met Val Ala Tyr Met Leu Glu 195 200 205 Arg Glu Leu Val Arg Lys Thr Arg Phe Leu Pro Val Ala Gly Gly Thr 210 215 220 Ser Ser Val Tyr Ile Glu Val Leu His Leu Thr Gln Gly Thr Cys Trp 225 230 235 240 Glu Gln Met Tyr Thr Pro Gly Gly Glu Val Arg Asn Asp Asp Ile Asp 245 250 255 Gln Ser Leu Ile Ile Ala Ala Arg Asn Ile Val Arg Arg Ala Thr Val 260 265 270 Ser Ala Asp Pro Leu Ala Ser Leu Leu Glu Met Cys His Ser Thr Gln 275 280 285 Ile Gly Gly Ile Arg Met Val Asp Ile Leu Lys Gln Asn Pro Thr Glu 290 295 300 Glu Gln Ala Val Asp Ile Cys Lys Ala Ala Met Gly Leu Arg Ile Ser 305 310 315 320 Ser Ser Phe Ser Phe Gly Gly Phe Thr Phe Lys Arg Thr Ser Gly Ser 325 330 335 Ser Val Lys Arg Glu Glu Glu Met Leu Thr Gly Asn Leu Gln Thr Leu 340 345 350 Lys Ile Arg Val His Glu Gly Tyr Glu Glu Phe Thr Met Val Gly Arg 355 360 365 Arg Ala Thr Ala Ile Leu Arg Lys Ala Thr Arg Arg Leu Ile Gln Leu 370 375 380 Ile Val Ser Gly Arg Asp Glu Gln Ser Ile Ala Glu Ala Ile Ile Val 385 390 395 400 Ala Met Val Phe Ser Gln Glu Asp Cys Met Ile Lys Ala Val Arg Gly 405 410 415 Asp Leu Asn Phe Val Asn Arg Ala Asn Gln Arg Leu Asn Pro Met His 420 425 430 Gln Leu Leu Arg His Phe Gln Lys Asp Ala Lys Val Leu Phe Gln Asn 435 440 445 Trp Gly Ile Glu Pro Ile Asp Asn Val Met Gly Met Ile Gly Ile Leu 450 455 460 Pro Asp Met Thr Pro Ser Thr Glu Met Ser Leu Arg Gly Val Arg Val 465 470 475 480 Ser Lys Met Gly Val Asp Glu Tyr Ser Ser Thr Glu Arg Val Val Val 485 490 495 Ser Ile Asp Arg Phe Leu Arg Val Arg Asp Gln Arg Gly Asn Ile Leu 500 505 510 Leu Ser Pro Glu Glu Val Ser Glu Thr Gln Gly Thr Glu Lys Leu Thr 515 520 525 Ile Ile Tyr Ser Ser Ser Met Met Trp Glu Ile Asn Gly Pro Glu Ser 530 535 540 Val Leu Val Asn Thr Tyr Gln Trp Ile Ile Arg Asn Trp Glu Ile Val 545 550 555 560 Lys Ile Gln Trp Ser Gln Asp Pro Thr Met Leu Tyr Asn Lys Ile Glu 565 570 575 Phe Glu Pro Phe Gln Ser Leu Val Pro Arg Ala Thr Arg Ser Gln Tyr 580 585 590 Ser Gly Phe Val Arg Thr Leu Phe Gln Gln Met Arg Asp Val Leu Gly 595 600 605 Thr Phe Asp Thr Ala Gln Ile Ile Lys Leu Leu Pro Phe Ala Ala Ala 610 615 620 Pro Pro Glu Gln Ser Arg Met Gln Phe Ser Ser Leu Thr Val Asn Val 625 630 635 640 Arg Gly Ser Gly Met Arg Ile Leu Val Arg Gly Asn Ser Pro Val Phe 645 650 655 Asn Tyr Asn Lys Ala Thr Lys Arg Leu Thr Val Leu Gly Lys Asp Ala 660 665 670 Gly Ala Leu Thr Glu Asp Pro Asp Glu Gly Thr Ala Gly Val Glu Ser 675 680 685 Ala Val Leu Arg Gly Phe Leu Ile Leu Gly Lys Glu Asn Lys Arg Tyr 690 695 700 Gly Pro Ala Leu Ser Ile Asn Glu Leu Ser Lys Leu Ala Lys Gly Glu 705 710 715 720 Lys Ala Asn Val Leu Ile Gly Gln Gly Asp Val Val Leu Val Met Lys 725 730 735 Arg Lys Arg Asp Ser Ser Ile Leu Thr Asp Ser Gln Thr Ala Thr Lys 740 745 750 Arg Ile Arg Met Ala Ile Asn 755 46 2277 DNA Equine influenza virus H3N8 46 atggagagaa taaaagaact gagagatcta atgtcacaat cccgcacccg cgagatacta 60 acaaaaacta ctgtggacca catggccata atcaagaaat acacatcagg aagacaagag 120 aagaaccccg cacttaggat gaagtggatg atggcaatga aatacccaat tacagcagat 180 aagaggataa tggaaatgat tcctgagaga aatgaacagg ggcaaaccct ttggagcaaa 240 acgaacgatg ctggctcaga ccgcgtaatg gtatcacctc tggcagtgac atggtggaat 300 aggaatggac caacaacgag cacaattcat tatccaaaag tctacaaaac ttattttgaa 360 aaagttgaaa gattaaaaca cggaaccttt ggccccgttc attttaggaa tcaagtcaag 420 ataagacgga gagttgatgt aaaccctggt cacgcggacc tcagtgccaa agaagcacaa 480 gatgtgatca tggaagttgt tttcccaaat gaagtgggag ccagaattct aacatcggaa 540 tcacaactaa caataaccaa agagaaaaaa gaagaacttc aggactgcaa aattgccccc 600 ttgatggtag catacatgct agaaagagag ttggtccgaa aaacaagatt cctcccagtg 660 gctggcggaa caagcagtgt atacattgaa gtgttgcatc tgactcaggg aacatgctgg 720 gaacaaatgt acaccccagg aggagaagtt agaaacgatg acattgatca aagtttaatt 780 attgctgccc ggaacatagt gagaagagcg acagtatcag cagatccact agcatccctg 840 ctggaaatgt gccacagtac acagattggt ggaataagga tggtagacat ccttaagcag 900 aatccaacag aggaacaagc tgtggatata tgcaaagcag caatggggtt aagaattagc 960 tcatcattca gctttggtgg attcaccttt aagagaacaa gtggatcatc agtcaagaga 1020 gaagaagaaa tgcttacggg caaccttcaa acattgaaaa taagagtgca tgaaggctat 1080 gaagaattca caatggtcgg aagaagagca acagccattc tcagaaaggc aaccagaaga 1140 ttgattcaat tgatagtaag tgggagagat gaacaatcaa ttgctgaagc aataattgta 1200 gccatggtgt tttcgcaaga agattgcatg ataaaagcag ttcgaggcga tttgaacttc 1260 gttaatagag caaatcagcg cttgaacccc atgcatcaac tcttgaggca tttccaaaaa 1320 gatgcaaaag tgcttttcca gaattggggg attgaaccca tcgacaatgt gatgggaatg 1380 attggaatat tgcctgacat gaccccaagc accgagatgt cattgagagg agtgagagtc 1440 agcaaaatgg gagtggatga gtactccagc actgagagag tggtggtgag cattgaccgt 1500 tttttaagag ttcgggatca aaggggaaac atactactgt cccctgaaga ggtcagtgaa 1560 acacaaggaa cggaaaagct gacaataatt tattcatcat caatgatgtg ggagattaat 1620 ggtcccgaat cagtgttggt caatacttat caatggatca tcaggaactg ggaaattgtg 1680 aaaattcaat ggtcacagga tcccacaatg ttatacaata agatagaatt tgagccattc 1740 cagtccctgg tccctagggc caccagaagc caatacagcg gtttcgtaag aaccctgttt 1800 cagcaaatgc gagatgtact tggaacattt gatactgctc aaataataaa actcctccct 1860 tttgccgctg ctcctccgga acagagtagg atgcagttct cttctttgac tgttaatgta 1920 agaggatcgg gaatgaggat acttgtaaga ggcaattccc cagtgttcaa ctacaataaa 1980 gccactaaga ggctcacagt cctcggaaag gatgcaggtg cgcttactga agacccagat 2040 gaaggtacgg ctggagtaga atctgctgtt ctaagagggt ttctcatttt aggtaaagaa 2100 aacaagagat atggcccagc actaagcatc aatgaactga gcaaacttgc aaaaggggag 2160 aaagctaatg tgctaattgg gcaaggggac gtggtgttgg taatgaaacg gaaacgtgac 2220 tctagcatac ttactgacag ccagacagcg accaaaagga ttcggatggc catcaat 2277 47 2341 DNA Equine influenza virus H3N8 CDS (28)..(2304) 47 agcaaaagca ggtcaaatat attcaat atg gag aga ata aaa gaa ctg aga gat 54 Met Glu Arg Ile Lys Glu Leu Arg Asp 1 5 cta atg tca caa tcc cgc acc cgc gag ata cta aca aaa act act gtg 102 Leu Met Ser Gln Ser Arg Thr Arg Glu Ile Leu Thr Lys Thr Thr Val 10 15 20 25 gac cac atg gcc ata atc aag aaa tac aca tca gga aga caa gag aag 150 Asp His Met Ala Ile Ile Lys Lys Tyr Thr Ser Gly Arg Gln Glu Lys 30 35 40 aac ccc gca ctt agg atg aag tgg atg atg gca atg aaa tac cca att 198 Asn Pro Ala Leu Arg Met Lys Trp Met Met Ala Met Lys Tyr Pro Ile 45 50 55 aca gca gat aag agg ata atg gaa atg att cct gag aga aat gaa cag 246 Thr Ala Asp Lys Arg Ile Met Glu Met Ile Pro Glu Arg Asn Glu Gln 60 65 70 ggg caa acc ctt tgg agc aaa acg aac gat gct ggc tca gac cgc gta 294 Gly Gln Thr Leu Trp Ser Lys Thr Asn Asp Ala Gly Ser Asp Arg Val 75 80 85 atg gta tca cct ctg gca gtg aca tgg tgg aat agg aat gga cca aca 342 Met Val Ser Pro Leu Ala Val Thr Trp Trp Asn Arg Asn Gly Pro Thr 90 95 100 105 acg agc aca att cat tat cca aaa gtc cac aaa act tat ttt gaa aaa 390 Thr Ser Thr Ile His Tyr Pro Lys Val His Lys Thr Tyr Phe Glu Lys 110 115 120 gtt gaa aga tta aaa cac gga acc ttt ggc ccc gtt cat ttt agg aat 438 Val Glu Arg Leu Lys His Gly Thr Phe Gly Pro Val His Phe Arg Asn 125 130 135 caa gtc aag ata aga cgg aga gtt gat gta aac cct ggt cac gcg gac 486 Gln Val Lys Ile Arg Arg Arg Val Asp Val Asn Pro Gly His Ala Asp 140 145 150 ctc agt gcc aaa gaa gca caa gat gtg atc atg gaa gtt gtt ttc cca 534 Leu Ser Ala Lys Glu Ala Gln Asp Val Ile Met Glu Val Val Phe Pro 155 160 165 aat gaa gtg gga gcc aga att cta aca tcg gaa tca caa cta aca ata 582 Asn Glu Val Gly Ala Arg Ile Leu Thr Ser Glu Ser Gln Leu Thr Ile 170 175 180 185 acc aaa gag aaa aaa gaa gaa ctt cag gac tgc aaa att gcc ccc ttg 630 Thr Lys Glu Lys Lys Glu Glu Leu Gln Asp Cys Lys Ile Ala Pro Leu 190 195 200 atg gta gca tac atg cta gaa aga gag ttg gtc cga aaa aca aga ttc 678 Met Val Ala Tyr Met Leu Glu Arg Glu Leu Val Arg Lys Thr Arg Phe 205 210 215 ctc cca gtg gct ggc gga aca agc agt gta tac att gaa gtg ttg cat 726 Leu Pro Val Ala Gly Gly Thr Ser Ser Val Tyr Ile Glu Val Leu His 220 225 230 ctg act cag gga aca tgc tgg gaa caa atg tac acc cca gga gga gaa 774 Leu Thr Gln Gly Thr Cys Trp Glu Gln Met Tyr Thr Pro Gly Gly Glu 235 240 245 gtt aga aac gat gac att gat caa agt tta att att gct gcc cgg aac 822 Val Arg Asn Asp Asp Ile Asp Gln Ser Leu Ile Ile Ala Ala Arg Asn 250 255 260 265 ata gtg aga aga gcg aca gta tca gca gat cca cta gca tcc ctg ctg 870 Ile Val Arg Arg Ala Thr Val Ser Ala Asp Pro Leu Ala Ser Leu Leu 270 275 280 gaa atg tgc cac agt aca cag att ggt gga ata agg atg gta gac atc 918 Glu Met Cys His Ser Thr Gln Ile Gly Gly Ile Arg Met Val Asp Ile 285 290 295 ctt aag cag aat cca aca gag gaa caa gct gtg gat ata tgc aaa gca 966 Leu Lys Gln Asn Pro Thr Glu Glu Gln Ala Val Asp Ile Cys Lys Ala 300 305 310 gca atg ggg tta aga att agc tca tca ttc agc ttt ggt gga ttc acc 1014 Ala Met Gly Leu Arg Ile Ser Ser Ser Phe Ser Phe Gly Gly Phe Thr 315 320 325 ttt aag aga aca agt gga tca tca gtc aag aga gaa gaa gaa atg ctt 1062 Phe Lys Arg Thr Ser Gly Ser Ser Val Lys Arg Glu Glu Glu Met Leu 330 335 340 345 acg ggc aac ctt caa aca ttg aaa ata aga gtg cat gaa ggc tat gaa 1110 Thr Gly Asn Leu Gln Thr Leu Lys Ile Arg Val His Glu Gly Tyr Glu 350 355 360 gaa ttc aca atg gtc gga aga aga gca aca gcc att ctc aga aag gca 1158 Glu Phe Thr Met Val Gly Arg Arg Ala Thr Ala Ile Leu Arg Lys Ala 365 370 375 acc aga aga ttg att caa ttg ata gta agt ggg aga gat gaa caa tca 1206 Thr Arg Arg Leu Ile Gln Leu Ile Val Ser Gly Arg Asp Glu Gln Ser 380 385 390 att gct gaa gca ata att gta gcc atg gtg ttt tcg caa gaa gat tgc 1254 Ile Ala Glu Ala Ile Ile Val Ala Met Val Phe Ser Gln Glu Asp Cys 395 400 405 atg ata caa gca gtt cga ggc gat ttg aac ttc gtt aat aga gca aat 1302 Met Ile Gln Ala Val Arg Gly Asp Leu Asn Phe Val Asn Arg Ala Asn 410 415 420 425 cag cgc ttg aac ccc atg cat caa ctc ttg agg cat ttc caa aaa gat 1350 Gln Arg Leu Asn Pro Met His Gln Leu Leu Arg His Phe Gln Lys Asp 430 435 440 gca aaa gtg ctt ttc cag aat tgg ggg att gaa ccc atc gac aat gtg 1398 Ala Lys Val Leu Phe Gln Asn Trp Gly Ile Glu Pro Ile Asp Asn Val 445 450 455 atg gga atg att gga ata ttg cct gac atg acc cca agc acc gag atg 1446 Met Gly Met Ile Gly Ile Leu Pro Asp Met Thr Pro Ser Thr Glu Met 460 465 470 tca ttg aga gga gtg aga gtc agc aaa atg gga gtg gat gag tac tcc 1494 Ser Leu Arg Gly Val Arg Val Ser Lys Met Gly Val Asp Glu Tyr Ser 475 480 485 agc act gag aga gtg gtg gtg agc att gac cgt ttt tta aga gtt cgg 1542 Ser Thr Glu Arg Val Val Val Ser Ile Asp Arg Phe Leu Arg Val Arg 490 495 500 505 gat caa agg gga aac ata cta ctg tcc cct gaa gag gtc agt gaa aca 1590 Asp Gln Arg Gly Asn Ile Leu Leu Ser Pro Glu Glu Val Ser Glu Thr 510 515 520 caa gga acg gaa aag ctg aca ata att tat tca tca tca atg atg tgg 1638 Gln Gly Thr Glu Lys Leu Thr Ile Ile Tyr Ser Ser Ser Met Met Trp 525 530 535 gag att aat ggt ccc gaa tca gtg ttg gtc aat act tat caa tgg atc 1686 Glu Ile Asn Gly Pro Glu Ser Val Leu Val Asn Thr Tyr Gln Trp Ile 540 545 550 atc agg aac tgg gaa att gtg aaa att caa tgg tca cag gat ccc aca 1734 Ile Arg Asn Trp Glu Ile Val Lys Ile Gln Trp Ser Gln Asp Pro Thr 555 560 565 atg tta tac aat aag ata gaa ttt gag cca ttc cag tcc ctg gtc cct 1782 Met Leu Tyr Asn Lys Ile Glu Phe Glu Pro Phe Gln Ser Leu Val Pro 570 575 580 585 agg gcc acc aga agc caa tac agc ggt ttc gta aga acc ctg ttt cag 1830 Arg Ala Thr Arg Ser Gln Tyr Ser Gly Phe Val Arg Thr Leu Phe Gln 590 595 600 caa atg cga gat gta ctt gga aca ttt gat act gct caa ata ata aaa 1878 Gln Met Arg Asp Val Leu Gly Thr Phe Asp Thr Ala Gln Ile Ile Lys 605 610 615 ctc ctc cct ttt gcc gct gct cct ccg gaa cag agt agg atg cag ttc 1926 Leu Leu Pro Phe Ala Ala Ala Pro Pro Glu Gln Ser Arg Met Gln Phe 620 625 630 tct tct ttg act gtt aat gta aga gga tcg gga atg agg ata ctt gta 1974 Ser Ser Leu Thr Val Asn Val Arg Gly Ser Gly Met Arg Ile Leu Val 635 640 645 aga ggc aat tcc cca gtg ttc aac tac aat aaa gcc act aag agg ctc 2022 Arg Gly Asn Ser Pro Val Phe Asn Tyr Asn Lys Ala Thr Lys Arg Leu 650 655 660 665 aca gtc ctc gga aaa gat gca ggt gcg ctt act gaa gac cca gat gaa 2070 Thr Val Leu Gly Lys Asp Ala Gly Ala Leu Thr Glu Asp Pro Asp Glu 670 675 680 ggt acg gct gga gta gaa tct gct gtt cta aga ggg ttt ctc att tta 2118 Gly Thr Ala Gly Val Glu Ser Ala Val Leu Arg Gly Phe Leu Ile Leu 685 690 695 ggt aaa gaa aac aag aga tat ggc cca gca cta agc atc aat gaa ctg 2166 Gly Lys Glu Asn Lys Arg Tyr Gly Pro Ala Leu Ser Ile Asn Glu Leu 700 705 710 agc aaa ctt gca aaa ggg gag aaa gct aat gtg cta att ggg caa ggg 2214 Ser Lys Leu Ala Lys Gly Glu Lys Ala Asn Val Leu Ile Gly Gln Gly 715 720 725 gac gtg gtg ttg gta atg aaa cgg aaa cgt gac tct agc ata ctt act 2262 Asp Val Val Leu Val Met Lys Arg Lys Arg Asp Ser Ser Ile Leu Thr 730 735 740 745 gac agc cag aca gcg acc aaa agg att cgg atg gcc atc aat 2304 Asp Ser Gln Thr Ala Thr Lys Arg Ile Arg Met Ala Ile Asn 750 755 tagtgttgaa ttgtttaaaa acgaccttgt ttctact 2341 48 759 PRT Equine influenza virus H3N8 48 Met Glu Arg Ile Lys Glu Leu Arg Asp Leu Met Ser Gln Ser Arg Thr 1 5 10 15 Arg Glu Ile Leu Thr Lys Thr Thr Val Asp His Met Ala Ile Ile Lys 20 25 30 Lys Tyr Thr Ser Gly Arg Gln Glu Lys Asn Pro Ala Leu Arg Met Lys 35 40 45 Trp Met Met Ala Met Lys Tyr Pro Ile Thr Ala Asp Lys Arg Ile Met 50 55 60 Glu Met Ile Pro Glu Arg Asn Glu Gln Gly Gln Thr Leu Trp Ser Lys 65 70 75 80 Thr Asn Asp Ala Gly Ser Asp Arg Val Met Val Ser Pro Leu Ala Val 85 90 95 Thr Trp Trp Asn Arg Asn Gly Pro Thr Thr Ser Thr Ile His Tyr Pro 100 105 110 Lys Val His Lys Thr Tyr Phe Glu Lys Val Glu Arg Leu Lys His Gly 115 120 125 Thr Phe Gly Pro Val His Phe Arg Asn Gln Val Lys Ile Arg Arg Arg 130 135 140 Val Asp Val Asn Pro Gly His Ala Asp Leu Ser Ala Lys Glu Ala Gln 145 150 155 160 Asp Val Ile Met Glu Val Val Phe Pro Asn Glu Val Gly Ala Arg Ile 165 170 175 Leu Thr Ser Glu Ser Gln Leu Thr Ile Thr Lys Glu Lys Lys Glu Glu 180 185 190 Leu Gln Asp Cys Lys Ile Ala Pro Leu Met Val Ala Tyr Met Leu Glu 195 200 205 Arg Glu Leu Val Arg Lys Thr Arg Phe Leu Pro Val Ala Gly Gly Thr 210 215 220 Ser Ser Val Tyr Ile Glu Val Leu His Leu Thr Gln Gly Thr Cys Trp 225 230 235 240 Glu Gln Met Tyr Thr Pro Gly Gly Glu Val Arg Asn Asp Asp Ile Asp 245 250 255 Gln Ser Leu Ile Ile Ala Ala Arg Asn Ile Val Arg Arg Ala Thr Val 260 265 270 Ser Ala Asp Pro Leu Ala Ser Leu Leu Glu Met Cys His Ser Thr Gln 275 280 285 Ile Gly Gly Ile Arg Met Val Asp Ile Leu Lys Gln Asn Pro Thr Glu 290 295 300 Glu Gln Ala Val Asp Ile Cys Lys Ala Ala Met Gly Leu Arg Ile Ser 305 310 315 320 Ser Ser Phe Ser Phe Gly Gly Phe Thr Phe Lys Arg Thr Ser Gly Ser 325 330 335 Ser Val Lys Arg Glu Glu Glu Met Leu Thr Gly Asn Leu Gln Thr Leu 340 345 350 Lys Ile Arg Val His Glu Gly Tyr Glu Glu Phe Thr Met Val Gly Arg 355 360 365 Arg Ala Thr Ala Ile Leu Arg Lys Ala Thr Arg Arg Leu Ile Gln Leu 370 375 380 Ile Val Ser Gly Arg Asp Glu Gln Ser Ile Ala Glu Ala Ile Ile Val 385 390 395 400 Ala Met Val Phe Ser Gln Glu Asp Cys Met Ile Gln Ala Val Arg Gly 405 410 415 Asp Leu Asn Phe Val Asn Arg Ala Asn Gln Arg Leu Asn Pro Met His 420 425 430 Gln Leu Leu Arg His Phe Gln Lys Asp Ala Lys Val Leu Phe Gln Asn 435 440 445 Trp Gly Ile Glu Pro Ile Asp Asn Val Met Gly Met Ile Gly Ile Leu 450 455 460 Pro Asp Met Thr Pro Ser Thr Glu Met Ser Leu Arg Gly Val Arg Val 465 470 475 480 Ser Lys Met Gly Val Asp Glu Tyr Ser Ser Thr Glu Arg Val Val Val 485 490 495 Ser Ile Asp Arg Phe Leu Arg Val Arg Asp Gln Arg Gly Asn Ile Leu 500 505 510 Leu Ser Pro Glu Glu Val Ser Glu Thr Gln Gly Thr Glu Lys Leu Thr 515 520 525 Ile Ile Tyr Ser Ser Ser Met Met Trp Glu Ile Asn Gly Pro Glu Ser 530 535 540 Val Leu Val Asn Thr Tyr Gln Trp Ile Ile Arg Asn Trp Glu Ile Val 545 550 555 560 Lys Ile Gln Trp Ser Gln Asp Pro Thr Met Leu Tyr Asn Lys Ile Glu 565 570 575 Phe Glu Pro Phe Gln Ser Leu Val Pro Arg Ala Thr Arg Ser Gln Tyr 580 585 590 Ser Gly Phe Val Arg Thr Leu Phe Gln Gln Met Arg Asp Val Leu Gly 595 600 605 Thr Phe Asp Thr Ala Gln Ile Ile Lys Leu Leu Pro Phe Ala Ala Ala 610 615 620 Pro Pro Glu Gln Ser Arg Met Gln Phe Ser Ser Leu Thr Val Asn Val 625 630 635 640 Arg Gly Ser Gly Met Arg Ile Leu Val Arg Gly Asn Ser Pro Val Phe 645 650 655 Asn Tyr Asn Lys Ala Thr Lys Arg Leu Thr Val Leu Gly Lys Asp Ala 660 665 670 Gly Ala Leu Thr Glu Asp Pro Asp Glu Gly Thr Ala Gly Val Glu Ser 675 680 685 Ala Val Leu Arg Gly Phe Leu Ile Leu Gly Lys Glu Asn Lys Arg Tyr 690 695 700 Gly Pro Ala Leu Ser Ile Asn Glu Leu Ser Lys Leu Ala Lys Gly Glu 705 710 715 720 Lys Ala Asn Val Leu Ile Gly Gln Gly Asp Val Val Leu Val Met Lys 725 730 735 Arg Lys Arg Asp Ser Ser Ile Leu Thr Asp Ser Gln Thr Ala Thr Lys 740 745 750 Arg Ile Arg Met Ala Ile Asn 755 49 2277 DNA Equine influenza virus H3N8 49 atggagagaa taaaagaact gagagatcta atgtcacaat cccgcacccg cgagatacta 60 acaaaaacta ctgtggacca catggccata atcaagaaat acacatcagg aagacaagag 120 aagaaccccg cacttaggat gaagtggatg atggcaatga aatacccaat tacagcagat 180 aagaggataa tggaaatgat tcctgagaga aatgaacagg ggcaaaccct ttggagcaaa 240 acgaacgatg ctggctcaga ccgcgtaatg gtatcacctc tggcagtgac atggtggaat 300 aggaatggac caacaacgag cacaattcat tatccaaaag tccacaaaac ttattttgaa 360 aaagttgaaa gattaaaaca cggaaccttt ggccccgttc attttaggaa tcaagtcaag 420 ataagacgga gagttgatgt aaaccctggt cacgcggacc tcagtgccaa agaagcacaa 480 gatgtgatca tggaagttgt tttcccaaat gaagtgggag ccagaattct aacatcggaa 540 tcacaactaa caataaccaa agagaaaaaa gaagaacttc aggactgcaa aattgccccc 600 ttgatggtag catacatgct agaaagagag ttggtccgaa aaacaagatt cctcccagtg 660 gctggcggaa caagcagtgt atacattgaa gtgttgcatc tgactcaggg aacatgctgg 720 gaacaaatgt acaccccagg aggagaagtt agaaacgatg acattgatca aagtttaatt 780 attgctgccc ggaacatagt gagaagagcg acagtatcag cagatccact agcatccctg 840 ctggaaatgt gccacagtac acagattggt ggaataagga tggtagacat ccttaagcag 900 aatccaacag aggaacaagc tgtggatata tgcaaagcag caatggggtt aagaattagc 960 tcatcattca gctttggtgg attcaccttt aagagaacaa gtggatcatc agtcaagaga 1020 gaagaagaaa tgcttacggg caaccttcaa acattgaaaa taagagtgca tgaaggctat 1080 gaagaattca caatggtcgg aagaagagca acagccattc tcagaaaggc aaccagaaga 1140 ttgattcaat tgatagtaag tgggagagat gaacaatcaa ttgctgaagc aataattgta 1200 gccatggtgt tttcgcaaga agattgcatg atacaagcag ttcgaggcga tttgaacttc 1260 gttaatagag caaatcagcg cttgaacccc atgcatcaac tcttgaggca tttccaaaaa 1320 gatgcaaaag tgcttttcca gaattggggg attgaaccca tcgacaatgt gatgggaatg 1380 attggaatat tgcctgacat gaccccaagc accgagatgt cattgagagg agtgagagtc 1440 agcaaaatgg gagtggatga gtactccagc actgagagag tggtggtgag cattgaccgt 1500 tttttaagag ttcgggatca aaggggaaac atactactgt cccctgaaga ggtcagtgaa 1560 acacaaggaa cggaaaagct gacaataatt tattcatcat caatgatgtg ggagattaat 1620 ggtcccgaat cagtgttggt caatacttat caatggatca tcaggaactg ggaaattgtg 1680 aaaattcaat ggtcacagga tcccacaatg ttatacaata agatagaatt tgagccattc 1740 cagtccctgg tccctagggc caccagaagc caatacagcg gtttcgtaag aaccctgttt 1800 cagcaaatgc gagatgtact tggaacattt gatactgctc aaataataaa actcctccct 1860 tttgccgctg ctcctccgga acagagtagg atgcagttct cttctttgac tgttaatgta 1920 agaggatcgg gaatgaggat acttgtaaga ggcaattccc cagtgttcaa ctacaataaa 1980 gccactaaga ggctcacagt cctcggaaaa gatgcaggtg cgcttactga agacccagat 2040 gaaggtacgg ctggagtaga atctgctgtt ctaagagggt ttctcatttt aggtaaagaa 2100 aacaagagat atggcccagc actaagcatc aatgaactga gcaaacttgc aaaaggggag 2160 aaagctaatg tgctaattgg gcaaggggac gtggtgttgg taatgaaacg gaaacgtgac 2220 tctagcatac ttactgacag ccagacagcg accaaaagga ttcggatggc catcaat 2277 50 891 DNA Equine influenza virus H3N8 CDS (27)..(716) 50 agcaaaagca gggtgacaaa aacata atg gat tcc aac act gtg tca agc ttt 53 Met Asp Ser Asn Thr Val Ser Ser Phe 1 5 cag gta gac tgt ttt ctt tgg cat gtc cgc aaa cga ttt gca gac caa 101 Gln Val Asp Cys Phe Leu Trp His Val Arg Lys Arg Phe Ala Asp Gln 10 15 20 25 gaa ctg ggt gat gcc cca ttc ctt gac cgg ctt cgc cga gac cag aag 149 Glu Leu Gly Asp Ala Pro Phe Leu Asp Arg Leu Arg Arg Asp Gln Lys 30 35 40 tcc cta aaa gga aga ggt agc act ctt ggt ctg gac atc gaa aca gcc 197 Ser Leu Lys Gly Arg Gly Ser Thr Leu Gly Leu Asp Ile Glu Thr Ala 45 50 55 act cgt gca gga aag cag ata gtg gag cag att ctg gaa gag gaa tca 245 Thr Arg Ala Gly Lys Gln Ile Val Glu Gln Ile Leu Glu Glu Glu Ser 60 65 70 gat gag gca ctt aaa atg acc att gcc tct gtt cct gct tca cgc tac 293 Asp Glu Ala Leu Lys Met Thr Ile Ala Ser Val Pro Ala Ser Arg Tyr 75 80 85 tta act gac atg act ctt gat gag atg tca aga gac tgg ttc atg ctc 341 Leu Thr Asp Met Thr Leu Asp Glu Met Ser Arg Asp Trp Phe Met Leu 90 95 100 105 atg ccc aag cag aaa gta aca ggc tcc cta tgt ata aga atg gac cag 389 Met Pro Lys Gln Lys Val Thr Gly Ser Leu Cys Ile Arg Met Asp Gln 110 115 120 gca atc atg gat aag aac atc ata ctt aaa gca aac ttt agt gtg att 437 Ala Ile Met Asp Lys Asn Ile Ile Leu Lys Ala Asn Phe Ser Val Ile 125 130 135 ttc gaa agg ctg gag aca cta ata cta ctt aga gcc ttc acc gaa gaa 485 Phe Glu Arg Leu Glu Thr Leu Ile Leu Leu Arg Ala Phe Thr Glu Glu 140 145 150 gga gca gtc gtt ggc gaa att tca cca ttg cct tct ctt cca gga cat 533 Gly Ala Val Val Gly Glu Ile Ser Pro Leu Pro Ser Leu Pro Gly His 155 160 165 act aat gag gat gtc aaa aat gca att ggg gtc ctc atc gga gga ctt 581 Thr Asn Glu Asp Val Lys Asn Ala Ile Gly Val Leu Ile Gly Gly Leu 170 175 180 185 aaa tgg aat gat aat acg gtt aga atc tct gaa act cta cag aga ttc 629 Lys Trp Asn Asp Asn Thr Val Arg Ile Ser Glu Thr Leu Gln Arg Phe 190 195 200 gct tgg aga agc agt cat gag aat ggg aga cct tca ttc cct cca aag 677 Ala Trp Arg Ser Ser His Glu Asn Gly Arg Pro Ser Phe Pro Pro Lys 205 210 215 cag aaa cga aaa atg gag aga aca att gag cca gaa gtt tgaagaaata 726 Gln Lys Arg Lys Met Glu Arg Thr Ile Glu Pro Glu Val 220 225 230 agatggttga ttgaagaagt gcgacataga ttgaaaaata cagaaaatag ttttgaacaa 786 ataacattta tgcaagcctt acaactattg cttgaagtag gacaagagat aagaactttc 846 tcgtttcagc ttatttaatg ataaaaaaca cccttgtttc tacta 891 51 230 PRT Equine influenza virus H3N8 51 Met Asp Ser Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp 1 5 10 15 His Val Arg Lys Arg Phe Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe 20 25 30 Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Lys Gly Arg Gly Ser 35 40 45 Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr Arg Ala Gly Lys Gln Ile 50 55 60 Val Glu Gln Ile Leu Glu Glu Glu Ser Asp Glu Ala Leu Lys Met Thr 65 70 75 80 Ile Ala Ser Val Pro Ala Ser Arg Tyr Leu Thr Asp Met Thr Leu Asp 85 90 95 Glu Met Ser Arg Asp Trp Phe Met Leu Met Pro Lys Gln Lys Val Thr 100 105 110 Gly Ser Leu Cys Ile Arg Met Asp Gln Ala Ile Met Asp Lys Asn Ile 115 120 125 Ile Leu Lys Ala Asn Phe Ser Val Ile Phe Glu Arg Leu Glu Thr Leu 130 135 140 Ile Leu Leu Arg Ala Phe Thr Glu Glu Gly Ala Val Val Gly Glu Ile 145 150 155 160 Ser Pro Leu Pro Ser Leu Pro Gly His Thr Asn Glu Asp Val Lys Asn 165 170 175 Ala Ile Gly Val Leu Ile Gly Gly Leu Lys Trp Asn Asp Asn Thr Val 180 185 190 Arg Ile Ser Glu Thr Leu Gln Arg Phe Ala Trp Arg Ser Ser His Glu 195 200 205 Asn Gly Arg Pro Ser Phe Pro Pro Lys Gln Lys Arg Lys Met Glu Arg 210 215 220 Thr Ile Glu Pro Glu Val 225 230 52 690 DNA Equine influenza virus H3N8 52 atggattcca acactgtgtc aagctttcag gtagactgtt ttctttggca tgtccgcaaa 60 cgatttgcag accaagaact gggtgatgcc ccattccttg accggcttcg ccgagaccag 120 aagtccctaa aaggaagagg tagcactctt ggtctggaca tcgaaacagc cactcgtgca 180 ggaaagcaga tagtggagca gattctggaa gaggaatcag atgaggcact taaaatgacc 240 attgcctctg ttcctgcttc acgctactta actgacatga ctcttgatga gatgtcaaga 300 gactggttca tgctcatgcc caagcagaaa gtaacaggct ccctatgtat aagaatggac 360 caggcaatca tggataagaa catcatactt aaagcaaact ttagtgtgat tttcgaaagg 420 ctggagacac taatactact tagagccttc accgaagaag gagcagtcgt tggcgaaatt 480 tcaccattgc cttctcttcc aggacatact aatgaggatg tcaaaaatgc aattggggtc 540 ctcatcggag gacttaaatg gaatgataat acggttagaa tctctgaaac tctacagaga 600 ttcgcttgga gaagcagtca tgagaatggg agaccttcat tccctccaaa gcagaaacga 660 aaaatggaga gaacaattga gccagaagtt 690 53 888 DNA Equine influenza virus H3N8 53 caaaagcagg gtgacaaaaa catgatggat tccaacactg tgtcaagctt tcaggtagac 60 tgttttcttt ggcatgtccg caaacgattt gcagaccaag aactgggtga tgccccattc 120 cttgaccggc ttcgccgaga ccagaagtcc ctaaaaggaa gaggtagcac tcttggtctg 180 gacatcgaaa cagccactcg tgcaggaaag cagatagtgg agcagattct ggaagaggaa 240 tcagatgagg cacttaaaat gaccattgcc tctgttcctg cttcacgcta cttaactgac 300 atgactcttg atgagatgtc aagagactgg ttcatgctca tgcccaagca gaaagtaaca 360 ggctccctat gtataagaat ggaccaggca atcatggata agaacatcat acttaaagca 420 aactttagtg tgattttcga aaggctggag acactaatac tacttagagc cttcaccgaa 480 gaaggagcag tcgttggcga aatttcacca ttgccttctc ttccaggaca tactaatgag 540 gatgtcaaaa atgcaattgg ggtcctcatc ggaggactta aatggaatga taatacggtt 600 agaatctctg aaactctaca gagattcgct tggagaagca gtcatgagaa tgggagacct 660 tcattccctc caaagcagaa acgaaaaatg gagagaacaa ttgagccaga agtttgaaga 720 aataagatgg ttgattgaag aagtgcgaca tagattgaaa aatacagaaa atagttttga 780 acaaataaca tttatgcaag ccttacaact attgcttgaa gtagaacaag agataagaac 840 tttctcgttt cagcttattt aatgataaaa aacacccttg tttctact 888 54 468 DNA Equine influenza virus H3N8 CDS (3)..(293) 54 ac ttt agt gtg att ttc gaa agg ctg gag aca cta ata cta ctt aga 47 Phe Ser Val Ile Phe Glu Arg Leu Glu Thr Leu Ile Leu Leu Arg 1 5 10 15 gcc ttc acc gaa gaa gga gca gtc gtt ggc gaa att tca cca ttg cct 95 Ala Phe Thr Glu Glu Gly Ala Val Val Gly Glu Ile Ser Pro Leu Pro 20 25 30 tct ctt cca gga cat act aat gag gat gtc aaa aat gca att ggg gtc 143 Ser Leu Pro Gly His Thr Asn Glu Asp Val Lys Asn Ala Ile Gly Val 35 40 45 ctc atc gga gga ctt aaa tgg aat gat aat acg gtt aga atc tct gaa 191 Leu Ile Gly Gly Leu Lys Trp Asn Asp Asn Thr Val Arg Ile Ser Glu 50 55 60 act cta cag aga ttc gct cgg aga agc agt cat gag aat ggg aga cct 239 Thr Leu Gln Arg Phe Ala Arg Arg Ser Ser His Glu Asn Gly Arg Pro 65 70 75 tca ttc cct cca aag cag aaa cga aaa atg gag aga aca att gag cca 287 Ser Phe Pro Pro Lys Gln Lys Arg Lys Met Glu Arg Thr Ile Glu Pro 80 85 90 95 gaa gtt tgaagaaata agatggttga ttgaagaagt gcgacataga ttgaaaaata 343 Glu Val cagaaaatag ttttgaacaa ataacattta tgcaagcctt acaactattg cttgaagtag 403 aacaagagat aagaactttc tcgtttcagc ttatttaatg ataaaaaaca cccttgtttc 463 tacta 468 55 97 PRT Equine influenza virus H3N8 55 Phe Ser Val Ile Phe Glu Arg Leu Glu Thr Leu Ile Leu Leu Arg Ala 1 5 10 15 Phe Thr Glu Glu Gly Ala Val Val Gly Glu Ile Ser Pro Leu Pro Ser 20 25 30 Leu Pro Gly His Thr Asn Glu Asp Val Lys Asn Ala Ile Gly Val Leu 35 40 45 Ile Gly Gly Leu Lys Trp Asn Asp Asn Thr Val Arg Ile Ser Glu Thr 50 55 60 Leu Gln Arg Phe Ala Arg Arg Ser Ser His Glu Asn Gly Arg Pro Ser 65 70 75 80 Phe Pro Pro Lys Gln Lys Arg Lys Met Glu Arg Thr Ile Glu Pro Glu 85 90 95 Val 56 293 DNA Equine influenza virus H3N8 56 actttagtgt gattttcgaa aggctggaga cactaatact acttagagcc ttcaccgaag 60 aaggagcagt cgttggcgaa atttcaccat tgccttctct tccaggacat actaatgagg 120 atgtcaaaaa tgcaattggg gtcctcatcg gaggacttaa atggaatgat aatacggtta 180 gaatctctga aactctacag agattcgctc ggagaagcag tcatgagaat gggagacctt 240 cattccctcc aaagcagaaa cgaaaaatgg agagaacaat tgagccagaa gtt 293 57 888 DNA Equine influenza virus H3N8 CDS (27)..(716) 57 agcaaaagca gggtgacaaa aacata atg gat tcc aac act gtg tca agc ttt 53 Met Asp Ser Asn Thr Val Ser Ser Phe 1 5 cag gta gac tgt ttt ctt tgg cat gtc cgc aaa cga ttt gca gac caa 101 Gln Val Asp Cys Phe Leu Trp His Val Arg Lys Arg Phe Ala Asp Gln 10 15 20 25 gaa ctg ggt gat gcc cca ttc ctt gac cgg ctt cgc cga gac cag aag 149 Glu Leu Gly Asp Ala Pro Phe Leu Asp Arg Leu Arg Arg Asp Gln Lys 30 35 40 tcc cta aaa gga aga ggt agc act ctt ggt ctg gac atc gaa aca gcc 197 Ser Leu Lys Gly Arg Gly Ser Thr Leu Gly Leu Asp Ile Glu Thr Ala 45 50 55 act cgt gca gga aag cag ata gtg gag cag att ctg gaa gag gaa tca 245 Thr Arg Ala Gly Lys Gln Ile Val Glu Gln Ile Leu Glu Glu Glu Ser 60 65 70 gat gag gca ctt aaa atg acc att gcc tct gtt cct gct tca cgc tac 293 Asp Glu Ala Leu Lys Met Thr Ile Ala Ser Val Pro Ala Ser Arg Tyr 75 80 85 tta act gac atg act ctt gat gag atg tca aga gac tgg ttc atg ctc 341 Leu Thr Asp Met Thr Leu Asp Glu Met Ser Arg Asp Trp Phe Met Leu 90 95 100 105 atg ccc aag cag aaa gta aca ggc tcc cta tgt ata aga atg gac cag 389 Met Pro Lys Gln Lys Val Thr Gly Ser Leu Cys Ile Arg Met Asp Gln 110 115 120 gca atc atg gat aag aac atc ata ctt aaa gca aac ttt agt gtg att 437 Ala Ile Met Asp Lys Asn Ile Ile Leu Lys Ala Asn Phe Ser Val Ile 125 130 135 ttc gaa agg ctg gag aca cta ata cta ctt aga gcc ttc acc gaa gaa 485 Phe Glu Arg Leu Glu Thr Leu Ile Leu Leu Arg Ala Phe Thr Glu Glu 140 145 150 gga gca gtc gtt ggc gaa att tca cca ttg cct tct ctt cca gga cat 533 Gly Ala Val Val Gly Glu Ile Ser Pro Leu Pro Ser Leu Pro Gly His 155 160 165 act aat gag gat gtc aaa aat gca att ggg gtc ctc atc gga gga ctt 581 Thr Asn Glu Asp Val Lys Asn Ala Ile Gly Val Leu Ile Gly Gly Leu 170 175 180 185 aaa tgg aat gat aat acg gtt aga atc tct gaa act cta cag aga ttc 629 Lys Trp Asn Asp Asn Thr Val Arg Ile Ser Glu Thr Leu Gln Arg Phe 190 195 200 gct tgg aga agc agt cat gag aat ggg aga cct tca ttc cct cca aag 677 Ala Trp Arg Ser Ser His Glu Asn Gly Arg Pro Ser Phe Pro Pro Lys 205 210 215 cag aaa cga aaa atg gag aga aca att gag cca gaa gtt tgaagaaata 726 Gln Lys Arg Lys Met Glu Arg Thr Ile Glu Pro Glu Val 220 225 230 agatggttga ttgaagaagt gcgacataga ttgaaaaata cagaaaatag ttttgaacaa 786 ataacattta tgcaagcctt acaactattg cttgaagtag aacaagagat aagaactttc 846 tcgtttcagc ttatttaatg ataaaaaaca cccttgtttc ta 888 58 230 PRT Equine influenza virus H3N8 58 Met Asp Ser Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp 1 5 10 15 His Val Arg Lys Arg Phe Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe 20 25 30 Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Lys Gly Arg Gly Ser 35 40 45 Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr Arg Ala Gly Lys Gln Ile 50 55 60 Val Glu Gln Ile Leu Glu Glu Glu Ser Asp Glu Ala Leu Lys Met Thr 65 70 75 80 Ile Ala Ser Val Pro Ala Ser Arg Tyr Leu Thr Asp Met Thr Leu Asp 85 90 95 Glu Met Ser Arg Asp Trp Phe Met Leu Met Pro Lys Gln Lys Val Thr 100 105 110 Gly Ser Leu Cys Ile Arg Met Asp Gln Ala Ile Met Asp Lys Asn Ile 115 120 125 Ile Leu Lys Ala Asn Phe Ser Val Ile Phe Glu Arg Leu Glu Thr Leu 130 135 140 Ile Leu Leu Arg Ala Phe Thr Glu Glu Gly Ala Val Val Gly Glu Ile 145 150 155 160 Ser Pro Leu Pro Ser Leu Pro Gly His Thr Asn Glu Asp Val Lys Asn 165 170 175 Ala Ile Gly Val Leu Ile Gly Gly Leu Lys Trp Asn Asp Asn Thr Val 180 185 190 Arg Ile Ser Glu Thr Leu Gln Arg Phe Ala Trp Arg Ser Ser His Glu 195 200 205 Asn Gly Arg Pro Ser Phe Pro Pro Lys Gln Lys Arg Lys Met Glu Arg 210 215 220 Thr Ile Glu Pro Glu Val 225 230 59 690 DNA Equine influenza virus H3N8 59 atggattcca acactgtgtc aagctttcag gtagactgtt ttctttggca tgtccgcaaa 60 cgatttgcag accaagaact gggtgatgcc ccattccttg accggcttcg ccgagaccag 120 aagtccctaa aaggaagagg tagcactctt ggtctggaca tcgaaacagc cactcgtgca 180 ggaaagcaga tagtggagca gattctggaa gaggaatcag atgaggcact taaaatgacc 240 attgcctctg ttcctgcttc acgctactta actgacatga ctcttgatga gatgtcaaga 300 gactggttca tgctcatgcc caagcagaaa gtaacaggct ccctatgtat aagaatggac 360 caggcaatca tggataagaa catcatactt aaagcaaact ttagtgtgat tttcgaaagg 420 ctggagacac taatactact tagagccttc accgaagaag gagcagtcgt tggcgaaatt 480 tcaccattgc cttctcttcc aggacatact aatgaggatg tcaaaaatgc aattggggtc 540 ctcatcggag gacttaaatg gaatgataat acggttagaa tctctgaaac tctacagaga 600 ttcgcttgga gaagcagtca tgagaatggg agaccttcat tccctccaaa gcagaaacga 660 aaaatggaga gaacaattga gccagaagtt 690 60 21 DNA Artificial sequence Synthetic Primer 60 agcaaagcag gtgacaaaaa c 21 61 19 DNA Artificial sequence Synthetic Primer 61 agtagaaaca agggtgttt 19 62 1229 DNA Equine influenza virus H3N8 CDS (36)..(1229) 62 gaattcggct tagcaaaagc aggcaaacta tttga atg gat gtc aat ccg act 53 Met Asp Val Asn Pro Thr 1 5 cta ctc ttc tta aag gtg cca gcg caa aat gct ata agc aca aca ttc 101 Leu Leu Phe Leu Lys Val Pro Ala Gln Asn Ala Ile Ser Thr Thr Phe 10 15 20 cct tat act gga gat cct ccc tac agt cat gga aca ggg aca gga tac 149 Pro Tyr Thr Gly Asp Pro Pro Tyr Ser His Gly Thr Gly Thr Gly Tyr 25 30 35 acc atg gat act gtc aac aga aca cat caa tac tca gaa aag ggg aaa 197 Thr Met Asp Thr Val Asn Arg Thr His Gln Tyr Ser Glu Lys Gly Lys 40 45 50 tgg aca aca aac act gag att gga gca cca caa ctt aat cca atc gat 245 Trp Thr Thr Asn Thr Glu Ile Gly Ala Pro Gln Leu Asn Pro Ile Asp 55 60 65 70 gga ccg ctt cct gaa gac aat gaa cca agt ggg tac gcc caa aca gat 293 Gly Pro Leu Pro Glu Asp Asn Glu Pro Ser Gly Tyr Ala Gln Thr Asp 75 80 85 tgt gta ttg gaa gca atg gct ttc ctt gaa gaa tcc cat ccc gga atc 341 Cys Val Leu Glu Ala Met Ala Phe Leu Glu Glu Ser His Pro Gly Ile 90 95 100 ttt gaa aat tcg tgt ctt gaa aca atg gag gtg gtt cag cag aca aga 389 Phe Glu Asn Ser Cys Leu Glu Thr Met Glu Val Val Gln Gln Thr Arg 105 110 115 gtg gac aaa cta aca caa ggc cga caa act tac gat tgg acc ttg aat 437 Val Asp Lys Leu Thr Gln Gly Arg Gln Thr Tyr Asp Trp Thr Leu Asn 120 125 130 agg aat caa cct gcc gca aca gca ctt gct aat aca att gaa gtg ttc 485 Arg Asn Gln Pro Ala Ala Thr Ala Leu Ala Asn Thr Ile Glu Val Phe 135 140 145 150 aga tca aat gat ctg act tcc agt gag tca ggg aga tta atg gac ttc 533 Arg Ser Asn Asp Leu Thr Ser Ser Glu Ser Gly Arg Leu Met Asp Phe 155 160 165 ctc aaa gat gtc atg gag tcc atg aac aag gaa gaa atg gaa ata aca 581 Leu Lys Asp Val Met Glu Ser Met Asn Lys Glu Glu Met Glu Ile Thr 170 175 180 aca cac ttc caa cgg aag aga aga gta aga gac aac atg aca aag aga 629 Thr His Phe Gln Arg Lys Arg Arg Val Arg Asp Asn Met Thr Lys Arg 185 190 195 atg gtg aca cag aga acc ata ggg aag aaa aaa caa cga tta aac aga 677 Met Val Thr Gln Arg Thr Ile Gly Lys Lys Lys Gln Arg Leu Asn Arg 200 205 210 aag agc tat ctg atc agg gca tta acc tta aac aca atg acc aag gac 725 Lys Ser Tyr Leu Ile Arg Ala Leu Thr Leu Asn Thr Met Thr Lys Asp 215 220 225 230 gct gag aga ggg aaa ttg aaa cga cga gca att gca acc cca gga atg 773 Ala Glu Arg Gly Lys Leu Lys Arg Arg Ala Ile Ala Thr Pro Gly Met 235 240 245 cag ata aga ggg ttt gta tat ttt gtt gaa aca tta gcc cga aga ata 821 Gln Ile Arg Gly Phe Val Tyr Phe Val Glu Thr Leu Ala Arg Arg Ile 250 255 260 tgt gaa aag ctt gaa caa tca gga ttg cca gtt ggc ggt aat gag aaa 869 Cys Glu Lys Leu Glu Gln Ser Gly Leu Pro Val Gly Gly Asn Glu Lys 265 270 275 aag gcc aaa ctg gct aat gtc gtc aga aaa atg atg act aat tcc caa 917 Lys Ala Lys Leu Ala Asn Val Val Arg Lys Met Met Thr Asn Ser Gln 280 285 290 gac act gaa ctc tcc ttc acc atc act ggg gac aat acc aaa tgg aat 965 Asp Thr Glu Leu Ser Phe Thr Ile Thr Gly Asp Asn Thr Lys Trp Asn 295 300 305 310 gaa aat cag aac cca cgc atg ttc ctg gca atg atc aca tac ata act 1013 Glu Asn Gln Asn Pro Arg Met Phe Leu Ala Met Ile Thr Tyr Ile Thr 315 320 325 aga aac cag cca gaa tgg ttc aga aat gtt cta agc att gca ccg att 1061 Arg Asn Gln Pro Glu Trp Phe Arg Asn Val Leu Ser Ile Ala Pro Ile 330 335 340 atg ttc tca aat aaa atg gca aga ctg ggg aaa gga tat atg ttt gaa 1109 Met Phe Ser Asn Lys Met Ala Arg Leu Gly Lys Gly Tyr Met Phe Glu 345 350 355 agc aaa agt atg aaa ttg aga act caa ata cca gca gaa atg ctc gca 1157 Ser Lys Ser Met Lys Leu Arg Thr Gln Ile Pro Ala Glu Met Leu Ala 360 365 370 agc att gat ctg aaa tat ttc aat gat tca aca aaa aag aaa att gag 1205 Ser Ile Asp Leu Lys Tyr Phe Asn Asp Ser Thr Lys Lys Lys Ile Glu 375 380 385 390 aag ata cga cca caa gcc gaa ttc 1229 Lys Ile Arg Pro Gln Ala Glu Phe 395 63 398 PRT Equine influenza virus H3N8 63 Met Asp Val Asn Pro Thr Leu Leu Phe Leu Lys Val Pro Ala Gln Asn 1 5 10 15 Ala Ile Ser Thr Thr Phe Pro Tyr Thr Gly Asp Pro Pro Tyr Ser His 20 25 30 Gly Thr Gly Thr Gly Tyr Thr Met Asp Thr Val Asn Arg Thr His Gln 35 40 45 Tyr Ser Glu Lys Gly Lys Trp Thr Thr Asn Thr Glu Ile Gly Ala Pro 50 55 60 Gln Leu Asn Pro Ile Asp Gly Pro Leu Pro Glu Asp Asn Glu Pro Ser 65 70 75 80 Gly Tyr Ala Gln Thr Asp Cys Val Leu Glu Ala Met Ala Phe Leu Glu 85 90 95 Glu Ser His Pro Gly Ile Phe Glu Asn Ser Cys Leu Glu Thr Met Glu 100 105 110 Val Val Gln Gln Thr Arg Val Asp Lys Leu Thr Gln Gly Arg Gln Thr 115 120 125 Tyr Asp Trp Thr Leu Asn Arg Asn Gln Pro Ala Ala Thr Ala Leu Ala 130 135 140 Asn Thr Ile Glu Val Phe Arg Ser Asn Asp Leu Thr Ser Ser Glu Ser 145 150 155 160 Gly Arg Leu Met Asp Phe Leu Lys Asp Val Met Glu Ser Met Asn Lys 165 170 175 Glu Glu Met Glu Ile Thr Thr His Phe Gln Arg Lys Arg Arg Val Arg 180 185 190 Asp Asn Met Thr Lys Arg Met Val Thr Gln Arg Thr Ile Gly Lys Lys 195 200 205 Lys Gln Arg Leu Asn Arg Lys Ser Tyr Leu Ile Arg Ala Leu Thr Leu 210 215 220 Asn Thr Met Thr Lys Asp Ala Glu Arg Gly Lys Leu Lys Arg Arg Ala 225 230 235 240 Ile Ala Thr Pro Gly Met Gln Ile Arg Gly Phe Val Tyr Phe Val Glu 245 250 255 Thr Leu Ala Arg Arg Ile Cys Glu Lys Leu Glu Gln Ser Gly Leu Pro 260 265 270 Val Gly Gly Asn Glu Lys Lys Ala Lys Leu Ala Asn Val Val Arg Lys 275 280 285 Met Met Thr Asn Ser Gln Asp Thr Glu Leu Ser Phe Thr Ile Thr Gly 290 295 300 Asp Asn Thr Lys Trp Asn Glu Asn Gln Asn Pro Arg Met Phe Leu Ala 305 310 315 320 Met Ile Thr Tyr Ile Thr Arg Asn Gln Pro Glu Trp Phe Arg Asn Val 325 330 335 Leu Ser Ile Ala Pro Ile Met Phe Ser Asn Lys Met Ala Arg Leu Gly 340 345 350 Lys Gly Tyr Met Phe Glu Ser Lys Ser Met Lys Leu Arg Thr Gln Ile 355 360 365 Pro Ala Glu Met Leu Ala Ser Ile Asp Leu Lys Tyr Phe Asn Asp Ser 370 375 380 Thr Lys Lys Lys Ile Glu Lys Ile Arg Pro Gln Ala Glu Phe 385 390 395 64 1194 DNA Equine influenza virus H3N8 64 atggatgtca atccgactct actcttctta aaggtgccag cgcaaaatgc tataagcaca 60 acattccctt atactggaga tcctccctac agtcatggaa cagggacagg atacaccatg 120 gatactgtca acagaacaca tcaatactca gaaaagggga aatggacaac aaacactgag 180 attggagcac cacaacttaa tccaatcgat ggaccgcttc ctgaagacaa tgaaccaagt 240 gggtacgccc aaacagattg tgtattggaa gcaatggctt tccttgaaga atcccatccc 300 ggaatctttg aaaattcgtg tcttgaaaca atggaggtgg ttcagcagac aagagtggac 360 aaactaacac aaggccgaca aacttacgat tggaccttga ataggaatca acctgccgca 420 acagcacttg ctaatacaat tgaagtgttc agatcaaatg atctgacttc cagtgagtca 480 gggagattaa tggacttcct caaagatgtc atggagtcca tgaacaagga agaaatggaa 540 ataacaacac acttccaacg gaagagaaga gtaagagaca acatgacaaa gagaatggtg 600 acacagagaa ccatagggaa gaaaaaacaa cgattaaaca gaaagagcta tctgatcagg 660 gcattaacct taaacacaat gaccaaggac gctgagagag ggaaattgaa acgacgagca 720 attgcaaccc caggaatgca gataagaggg tttgtatatt ttgttgaaac attagcccga 780 agaatatgtg aaaagcttga acaatcagga ttgccagttg gcggtaatga gaaaaaggcc 840 aaactggcta atgtcgtcag aaaaatgatg actaattccc aagacactga actctccttc 900 accatcactg gggacaatac caaatggaat gaaaatcaga acccacgcat gttcctggca 960 atgatcacat acataactag aaaccagcca gaatggttca gaaatgttct aagcattgca 1020 ccgattatgt tctcaaataa aatggcaaga ctggggaaag gatatatgtt tgaaagcaaa 1080 agtatgaaat tgagaactca aataccagca gaaatgctcg caagcattga tctgaaatat 1140 ttcaatgatt caacaaaaaa gaaaattgag aagatacgac cacaagccga attc 1194 65 673 DNA Equine influenza virus H3N8 CDS (36)..(671) 65 gaattcggct tagcaaaagc aggcaaacta tttga atg gat gtc aat ccg act 53 Met Asp Val Asn Pro Thr 1 5 cta ctc ttc tta aag gtg cca gcg caa aat gct ata agc aca aca ttc 101 Leu Leu Phe Leu Lys Val Pro Ala Gln Asn Ala Ile Ser Thr Thr Phe 10 15 20 cct tat act gga gat cct ccc tac agt cat gga aca ggg aca gga tac 149 Pro Tyr Thr Gly Asp Pro Pro Tyr Ser His Gly Thr Gly Thr Gly Tyr 25 30 35 acc atg gat act gtc aac aga aca cat caa tac tca gaa aag ggg aaa 197 Thr Met Asp Thr Val Asn Arg Thr His Gln Tyr Ser Glu Lys Gly Lys 40 45 50 tgg aca aca aac act gag att gga gca cca caa ctt aat cca atc gat 245 Trp Thr Thr Asn Thr Glu Ile Gly Ala Pro Gln Leu Asn Pro Ile Asp 55 60 65 70 gga ccg ctt cct gaa gac aat gaa cca agt ggg tac gcc caa aca gat 293 Gly Pro Leu Pro Glu Asp Asn Glu Pro Ser Gly Tyr Ala Gln Thr Asp 75 80 85 tgt gta ttg gaa gca atg gct ttc ctt gaa gaa tcc cat ccc gga atc 341 Cys Val Leu Glu Ala Met Ala Phe Leu Glu Glu Ser His Pro Gly Ile 90 95 100 ttt gaa aat tcg tgt ctt gaa aca atg gag gtg gtt cag cag aca aga 389 Phe Glu Asn Ser Cys Leu Glu Thr Met Glu Val Val Gln Gln Thr Arg 105 110 115 gtg gac aaa cta aca caa ggc cga caa act tac gat tgg acc ttg aat 437 Val Asp Lys Leu Thr Gln Gly Arg Gln Thr Tyr Asp Trp Thr Leu Asn 120 125 130 agg aat caa cct gcc gca aca gca ctt gct aat aca att gaa gtg ttc 485 Arg Asn Gln Pro Ala Ala Thr Ala Leu Ala Asn Thr Ile Glu Val Phe 135 140 145 150 aga tca aat gat ctg act tcc agt gag tca ggg aga tta atg gac ttc 533 Arg Ser Asn Asp Leu Thr Ser Ser Glu Ser Gly Arg Leu Met Asp Phe 155 160 165 ctc aaa gat gtc atg gag tcc atg aac aag gaa gaa atg gaa ata aca 581 Leu Lys Asp Val Met Glu Ser Met Asn Lys Glu Glu Met Glu Ile Thr 170 175 180 aca cac ttc caa cgg aag aga aga gta aga gac aac atg aca aag aga 629 Thr His Phe Gln Arg Lys Arg Arg Val Arg Asp Asn Met Thr Lys Arg 185 190 195 atg gtg aca cag aga acc ata ggg aag aaa aaa caa cga tta aa 673 Met Val Thr Gln Arg Thr Ile Gly Lys Lys Lys Gln Arg Leu 200 205 210 66 212 PRT Equine influenza virus H3N8 66 Met Asp Val Asn Pro Thr Leu Leu Phe Leu Lys Val Pro Ala Gln Asn 1 5 10 15 Ala Ile Ser Thr Thr Phe Pro Tyr Thr Gly Asp Pro Pro Tyr Ser His 20 25 30 Gly Thr Gly Thr Gly Tyr Thr Met Asp Thr Val Asn Arg Thr His Gln 35 40 45 Tyr Ser Glu Lys Gly Lys Trp Thr Thr Asn Thr Glu Ile Gly Ala Pro 50 55 60 Gln Leu Asn Pro Ile Asp Gly Pro Leu Pro Glu Asp Asn Glu Pro Ser 65 70 75 80 Gly Tyr Ala Gln Thr Asp Cys Val Leu Glu Ala Met Ala Phe Leu Glu 85 90 95 Glu Ser His Pro Gly Ile Phe Glu Asn Ser Cys Leu Glu Thr Met Glu 100 105 110 Val Val Gln Gln Thr Arg Val Asp Lys Leu Thr Gln Gly Arg Gln Thr 115 120 125 Tyr Asp Trp Thr Leu Asn Arg Asn Gln Pro Ala Ala Thr Ala Leu Ala 130 135 140 Asn Thr Ile Glu Val Phe Arg Ser Asn Asp Leu Thr Ser Ser Glu Ser 145 150 155 160 Gly Arg Leu Met Asp Phe Leu Lys Asp Val Met Glu Ser Met Asn Lys 165 170 175 Glu Glu Met Glu Ile Thr Thr His Phe Gln Arg Lys Arg Arg Val Arg 180 185 190 Asp Asn Met Thr Lys Arg Met Val Thr Gln Arg Thr Ile Gly Lys Lys 195 200 205 Lys Gln Arg Leu 210 67 636 DNA Equine influenza virus H3N8 67 atggatgtca atccgactct actcttctta aaggtgccag cgcaaaatgc tataagcaca 60 acattccctt atactggaga tcctccctac agtcatggaa cagggacagg atacaccatg 120 gatactgtca acagaacaca tcaatactca gaaaagggga aatggacaac aaacactgag 180 attggagcac cacaacttaa tccaatcgat ggaccgcttc ctgaagacaa tgaaccaagt 240 gggtacgccc aaacagattg tgtattggaa gcaatggctt tccttgaaga atcccatccc 300 ggaatctttg aaaattcgtg tcttgaaaca atggaggtgg ttcagcagac aagagtggac 360 aaactaacac aaggccgaca aacttacgat tggaccttga ataggaatca acctgccgca 420 acagcacttg ctaatacaat tgaagtgttc agatcaaatg atctgacttc cagtgagtca 480 gggagattaa tggacttcct caaagatgtc atggagtcca tgaacaagga agaaatggaa 540 ataacaacac acttccaacg gaagagaaga gtaagagaca acatgacaaa gagaatggtg 600 acacagagaa ccatagggaa gaaaaaacaa cgatta 636 68 1225 DNA Equine influenza virus H3N8 CDS (34)..(1218) 68 gaattcagga gcaaaagcag gcaaactatt tga atg gat gtc aat ccg act cta 54 Met Asp Val Asn Pro Thr Leu 1 5 ctc ttc tta aag gtg cca gcg caa aat gct ata agc aca aca ttc cct 102 Leu Phe Leu Lys Val Pro Ala Gln Asn Ala Ile Ser Thr Thr Phe Pro 10 15 20 tat act gga gat cct ccc tac agt cat gga aca ggg aca gga tac acc 150 Tyr Thr Gly Asp Pro Pro Tyr Ser His Gly Thr Gly Thr Gly Tyr Thr 25 30 35 atg gat act gtc aac aga aca cat caa tac tca gaa aag ggg aaa tgg 198 Met Asp Thr Val Asn Arg Thr His Gln Tyr Ser Glu Lys Gly Lys Trp 40 45 50 55 aca aca aac act gag att gga gca cca caa ctt aat cca atc gat gga 246 Thr Thr Asn Thr Glu Ile Gly Ala Pro Gln Leu Asn Pro Ile Asp Gly 60 65 70 ccg ctt cct gaa gac aat gaa cca agt ggg tac gcc caa aca gat tgt 294 Pro Leu Pro Glu Asp Asn Glu Pro Ser Gly Tyr Ala Gln Thr Asp Cys 75 80 85 gta ttg gaa gca atg gct ttc ctt gaa gaa tcc cat ccc gga atc ttt 342 Val Leu Glu Ala Met Ala Phe Leu Glu Glu Ser His Pro Gly Ile Phe 90 95 100 gaa aat tcg tgt ctt gaa aca atg gag gtg gtt cag cag aca aga gtg 390 Glu Asn Ser Cys Leu Glu Thr Met Glu Val Val Gln Gln Thr Arg Val 105 110 115 gac aaa cta aca caa ggc cga caa act tac gat tgg acc ttg aat agg 438 Asp Lys Leu Thr Gln Gly Arg Gln Thr Tyr Asp Trp Thr Leu Asn Arg 120 125 130 135 aat caa cct gcc gca aca gca ctt gct aat aca att gaa gtg ttc aga 486 Asn Gln Pro Ala Ala Thr Ala Leu Ala Asn Thr Ile Glu Val Phe Arg 140 145 150 tca aat gat ctg act tcc agt gag tca ggg aga tta atg gac ttc ctc 534 Ser Asn Asp Leu Thr Ser Ser Glu Ser Gly Arg Leu Met Asp Phe Leu 155 160 165 aaa gat gtc atg gag tcc atg aac aag gaa gaa atg gaa ata aca aca 582 Lys Asp Val Met Glu Ser Met Asn Lys Glu Glu Met Glu Ile Thr Thr 170 175 180 cac ttc caa cgg aag aga aga gta aga gac aac atg aca aag aga atg 630 His Phe Gln Arg Lys Arg Arg Val Arg Asp Asn Met Thr Lys Arg Met 185 190 195 gtg aca cag aga acc ata ggg aag aaa aaa caa cga tta aac aga aag 678 Val Thr Gln Arg Thr Ile Gly Lys Lys Lys Gln Arg Leu Asn Arg Lys 200 205 210 215 agc tat ctg atc agg gca tta acc tta aac aca atg acc aag gac gct 726 Ser Tyr Leu Ile Arg Ala Leu Thr Leu Asn Thr Met Thr Lys Asp Ala 220 225 230 gag aga ggg aaa ttg aaa cga cga gca att gca acc cca gga atg cag 774 Glu Arg Gly Lys Leu Lys Arg Arg Ala Ile Ala Thr Pro Gly Met Gln 235 240 245 ata aga ggg ttt gta tat ttt gtt gaa aca tta gcc cga aga ata tgt 822 Ile Arg Gly Phe Val Tyr Phe Val Glu Thr Leu Ala Arg Arg Ile Cys 250 255 260 gaa aag ctt gaa caa tca gga ttg cca gtt ggc ggt aat gag aaa aag 870 Glu Lys Leu Glu Gln Ser Gly Leu Pro Val Gly Gly Asn Glu Lys Lys 265 270 275 gcc aaa ctg gct aat gtc gtc aga aaa atg atg act aat tcc caa gac 918 Ala Lys Leu Ala Asn Val Val Arg Lys Met Met Thr Asn Ser Gln Asp 280 285 290 295 act gaa ctc tcc ttc acc atc act ggg gac aat acc aaa tgg aat gaa 966 Thr Glu Leu Ser Phe Thr Ile Thr Gly Asp Asn Thr Lys Trp Asn Glu 300 305 310 aat cag aac cca cgc atg ttc ctg gca atg atc aca tac ata act aga 1014 Asn Gln Asn Pro Arg Met Phe Leu Ala Met Ile Thr Tyr Ile Thr Arg 315 320 325 aac cag cca gaa tgg ttc aga aat gtt cta agc att gca ccg att atg 1062 Asn Gln Pro Glu Trp Phe Arg Asn Val Leu Ser Ile Ala Pro Ile Met 330 335 340 ttc tca aat aaa atg gca aga ctg ggg aaa gga tat atg ttt gaa agc 1110 Phe Ser Asn Lys Met Ala Arg Leu Gly Lys Gly Tyr Met Phe Glu Ser 345 350 355 aaa agt atg aaa ttg aga act caa ata cca gca gaa atg ctc gca agc 1158 Lys Ser Met Lys Leu Arg Thr Gln Ile Pro Ala Glu Met Leu Ala Ser 360 365 370 375 att gat ctg aaa tat ttc aat gat tca aca aaa aag aaa att gag aag 1206 Ile Asp Leu Lys Tyr Phe Asn Asp Ser Thr Lys Lys Lys Ile Glu Lys 380 385 390 ata cga cca ccc tgaattc 1225 Ile Arg Pro Pro 395 69 395 PRT Equine influenza virus H3N8 69 Met Asp Val Asn Pro Thr Leu Leu Phe Leu Lys Val Pro Ala Gln Asn 1 5 10 15 Ala Ile Ser Thr Thr Phe Pro Tyr Thr Gly Asp Pro Pro Tyr Ser His 20 25 30 Gly Thr Gly Thr Gly Tyr Thr Met Asp Thr Val Asn Arg Thr His Gln 35 40 45 Tyr Ser Glu Lys Gly Lys Trp Thr Thr Asn Thr Glu Ile Gly Ala Pro 50 55 60 Gln Leu Asn Pro Ile Asp Gly Pro Leu Pro Glu Asp Asn Glu Pro Ser 65 70 75 80 Gly Tyr Ala Gln Thr Asp Cys Val Leu Glu Ala Met Ala Phe Leu Glu 85 90 95 Glu Ser His Pro Gly Ile Phe Glu Asn Ser Cys Leu Glu Thr Met Glu 100 105 110 Val Val Gln Gln Thr Arg Val Asp Lys Leu Thr Gln Gly Arg Gln Thr 115 120 125 Tyr Asp Trp Thr Leu Asn Arg Asn Gln Pro Ala Ala Thr Ala Leu Ala 130 135 140 Asn Thr Ile Glu Val Phe Arg Ser Asn Asp Leu Thr Ser Ser Glu Ser 145 150 155 160 Gly Arg Leu Met Asp Phe Leu Lys Asp Val Met Glu Ser Met Asn Lys 165 170 175 Glu Glu Met Glu Ile Thr Thr His Phe Gln Arg Lys Arg Arg Val Arg 180 185 190 Asp Asn Met Thr Lys Arg Met Val Thr Gln Arg Thr Ile Gly Lys Lys 195 200 205 Lys Gln Arg Leu Asn Arg Lys Ser Tyr Leu Ile Arg Ala Leu Thr Leu 210 215 220 Asn Thr Met Thr Lys Asp Ala Glu Arg Gly Lys Leu Lys Arg Arg Ala 225 230 235 240 Ile Ala Thr Pro Gly Met Gln Ile Arg Gly Phe Val Tyr Phe Val Glu 245 250 255 Thr Leu Ala Arg Arg Ile Cys Glu Lys Leu Glu Gln Ser Gly Leu Pro 260 265 270 Val Gly Gly Asn Glu Lys Lys Ala Lys Leu Ala Asn Val Val Arg Lys 275 280 285 Met Met Thr Asn Ser Gln Asp Thr Glu Leu Ser Phe Thr Ile Thr Gly 290 295 300 Asp Asn Thr Lys Trp Asn Glu Asn Gln Asn Pro Arg Met Phe Leu Ala 305 310 315 320 Met Ile Thr Tyr Ile Thr Arg Asn Gln Pro Glu Trp Phe Arg Asn Val 325 330 335 Leu Ser Ile Ala Pro Ile Met Phe Ser Asn Lys Met Ala Arg Leu Gly 340 345 350 Lys Gly Tyr Met Phe Glu Ser Lys Ser Met Lys Leu Arg Thr Gln Ile 355 360 365 Pro Ala Glu Met Leu Ala Ser Ile Asp Leu Lys Tyr Phe Asn Asp Ser 370 375 380 Thr Lys Lys Lys Ile Glu Lys Ile Arg Pro Pro 385 390 395 70 1185 DNA Equine influenza virus H3N8 70 atggatgtca atccgactct actcttctta aaggtgccag cgcaaaatgc tataagcaca 60 acattccctt atactggaga tcctccctac agtcatggaa cagggacagg atacaccatg 120 gatactgtca acagaacaca tcaatactca gaaaagggga aatggacaac aaacactgag 180 attggagcac cacaacttaa tccaatcgat ggaccgcttc ctgaagacaa tgaaccaagt 240 gggtacgccc aaacagattg tgtattggaa gcaatggctt tccttgaaga atcccatccc 300 ggaatctttg aaaattcgtg tcttgaaaca atggaggtgg ttcagcagac aagagtggac 360 aaactaacac aaggccgaca aacttacgat tggaccttga ataggaatca acctgccgca 420 acagcacttg ctaatacaat tgaagtgttc agatcaaatg atctgacttc cagtgagtca 480 gggagattaa tggacttcct caaagatgtc atggagtcca tgaacaagga agaaatggaa 540 ataacaacac acttccaacg gaagagaaga gtaagagaca acatgacaaa gagaatggtg 600 acacagagaa ccatagggaa gaaaaaacaa cgattaaaca gaaagagcta tctgatcagg 660 gcattaacct taaacacaat gaccaaggac gctgagagag ggaaattgaa acgacgagca 720 attgcaaccc caggaatgca gataagaggg tttgtatatt ttgttgaaac attagcccga 780 agaatatgtg aaaagcttga acaatcagga ttgccagttg gcggtaatga gaaaaaggcc 840 aaactggcta atgtcgtcag aaaaatgatg actaattccc aagacactga actctccttc 900 accatcactg gggacaatac caaatggaat gaaaatcaga acccacgcat gttcctggca 960 atgatcacat acataactag aaaccagcca gaatggttca gaaatgttct aagcattgca 1020 ccgattatgt tctcaaataa aatggcaaga ctggggaaag gatatatgtt tgaaagcaaa 1080 agtatgaaat tgagaactca aataccagca gaaatgctcg caagcattga tctgaaatat 1140 ttcaatgatt caacaaaaaa gaaaattgag aagatacgac caccc 1185 71 1221 DNA Equine influenza virus H3N8 71 gaattcagga aagcaggcaa actatttgaa tggatgtcaa tccgactcta ctcttcttaa 60 aggtgccagc gcaaaatgct ataagcacaa cattccctta tactggagat cctccctaca 120 gtcatggaac agggacagga tacaccatgg atactgtcaa cagaacacat caatactcag 180 aaaaggggaa atggacaaca aacactgaga ttggagcacc acaacttaat ccaatcgatg 240 gaccgcttcc tgaagacaat gaaccaagtg ggtacgccca aacagattgt gtattggaag 300 caatggcttt ccttgaagaa tcccatcccg gaatctttga aaattcgtgt cttgaaacaa 360 tggaggtggt tcagcagaca agagtggaca aactaacaca aggccgacaa acttacgatt 420 ggaccttgaa taggaatcaa cctgccgcaa cagcacttgc taatacaatt gaagtgttca 480 gatcaaatga tctgacttcc agtgagtcag ggagattaat ggacttcctc aaagatgtca 540 tggagtccat gaacaaggaa gaaatggaaa taacaacaca cttccaacgg aagagaagag 600 taagagacaa catgacaaag agaatggtga cacagagaac catagggaag aaaaaacaac 660 gattaaacag aaagagctat ctgatcaggg cattaacctt aaacacaatg accaaggacg 720 ctgagagagg gaaattgaaa cgacgagcaa ttgcaacccc aggaatgcag ataagagggt 780 ttgtatattt tgttgaaaca ttagcccgaa gaatatgtga aaagcttgaa caatcaggat 840 tgccagttgg cggtaatgag aaaaaggcca aactggctaa tgtcgtcaga aaaatgatga 900 ctaattccca agacactgaa ctctccttca ccatcactgg ggacaatacc aaatggaatg 960 aaaatcagaa cccacgcatg ttcctggcaa tgatcacata cataactaga aaccagccag 1020 aatggttcag aaatgttcta agcattgcac cgattatgtt ctcaaataaa atggcaagac 1080 tggggaaagg atatatgttt gaaagcaaaa gtatgaaatt gagaactcaa ataccagcag 1140 aaatgctcgc aagcattgat ctgaaatatt tcaatgattc aacaaaaaag aaaattgaga 1200 agatacgacc accctgaatt c 1221 72 18 DNA Artificial sequence Synthetic Primer 72 gcaaatgcag gaccaaag 18 73 18 DNA Artificial sequence Synthetic Primer 73 gactgaggac tcagcttc 18 74 19 DNA Artificial sequence Synthetic Primer 74 caatatcctc cccaatttc 19 75 19 DNA Artificial sequence Synthetic Primer 75 ggaaggtttg caggacctt 19 76 1228 DNA Equine influenza virus H3N8 CDS (3)..(1166) 76 gg ggc ggg tac cca aac tat ctc caa gct tgg aag caa gta tta gca 47 Gly Gly Tyr Pro Asn Tyr Leu Gln Ala Trp Lys Gln Val Leu Ala 1 5 10 15 gaa cta caa gac ctt gag aac gaa gaa aag acc cct aag acc aag aat 95 Glu Leu Gln Asp Leu Glu Asn Glu Glu Lys Thr Pro Lys Thr Lys Asn 20 25 30 atg aaa aaa aca agc caa ttg aaa tgg gca ctc ggt gaa aat atg gca 143 Met Lys Lys Thr Ser Gln Leu Lys Trp Ala Leu Gly Glu Asn Met Ala 35 40 45 cca gag aaa gtg gat ttt gag gat tgt aaa gac atc aat gat ttg aaa 191 Pro Glu Lys Val Asp Phe Glu Asp Cys Lys Asp Ile Asn Asp Leu Lys 50 55 60 cag tat gac agt gat gag cca gaa aca agg tct ctt gca agt tgg att 239 Gln Tyr Asp Ser Asp Glu Pro Glu Thr Arg Ser Leu Ala Ser Trp Ile 65 70 75 caa agt gag ttc aac aaa gct tgt gag ctg aca gat tca agc tgg ata 287 Gln Ser Glu Phe Asn Lys Ala Cys Glu Leu Thr Asp Ser Ser Trp Ile 80 85 90 95 gag ctc gat gaa att ggg gag gat att gcc cca ata gaa tac att gcg 335 Glu Leu Asp Glu Ile Gly Glu Asp Ile Ala Pro Ile Glu Tyr Ile Ala 100 105 110 agc atg agg aga aat tat ttt act gct gag gtt tcc cat tgt aga gca 383 Ser Met Arg Arg Asn Tyr Phe Thr Ala Glu Val Ser His Cys Arg Ala 115 120 125 aca gaa tat ata atg aag gga gtg tac atc aac act gct cta ctc aat 431 Thr Glu Tyr Ile Met Lys Gly Val Tyr Ile Asn Thr Ala Leu Leu Asn 130 135 140 gca tcc tgt gct gcg atg gat gaa ttc caa tta att ccg atg ata agc 479 Ala Ser Cys Ala Ala Met Asp Glu Phe Gln Leu Ile Pro Met Ile Ser 145 150 155 aaa tgc agg acc aaa gaa ggg aga agg aag aca aat tta tat gga ttc 527 Lys Cys Arg Thr Lys Glu Gly Arg Arg Lys Thr Asn Leu Tyr Gly Phe 160 165 170 175 ata ata aag gga agg tcc cat tta agg aat gat acc gac gtg gta aac 575 Ile Ile Lys Gly Arg Ser His Leu Arg Asn Asp Thr Asp Val Val Asn 180 185 190 ttt gta agt atg gaa ttt tct ctc act gat cca aga ttt gag cca cat 623 Phe Val Ser Met Glu Phe Ser Leu Thr Asp Pro Arg Phe Glu Pro His 195 200 205 aaa tgg gaa aaa tac tgc gtt cta gaa att gga gac atg ctc cta agg 671 Lys Trp Glu Lys Tyr Cys Val Leu Glu Ile Gly Asp Met Leu Leu Arg 210 215 220 act gct gta ggt caa gtg tca aga ccc atg ttt ttg tat gta agg aca 719 Thr Ala Val Gly Gln Val Ser Arg Pro Met Phe Leu Tyr Val Arg Thr 225 230 235 aat gga acc tct aaa att aaa atg aaa cgg gga atg gaa atg aga cgc 767 Asn Gly Thr Ser Lys Ile Lys Met Lys Arg Gly Met Glu Met Arg Arg 240 245 250 255 tgc ctc ctt cag tct ctg caa cag att gaa agc atg atc gaa gct gag 815 Cys Leu Leu Gln Ser Leu Gln Gln Ile Glu Ser Met Ile Glu Ala Glu 260 265 270 tcc tca gtc aaa gaa aag gac atg acc aaa gaa ttc ttt gag aac aaa 863 Ser Ser Val Lys Glu Lys Asp Met Thr Lys Glu Phe Phe Glu Asn Lys 275 280 285 tca gag aca tgg cct ata gga gag tcc ccc aaa gga gtg gaa gag ggc 911 Ser Glu Thr Trp Pro Ile Gly Glu Ser Pro Lys Gly Val Glu Glu Gly 290 295 300 tca atc ggg aag gtt tgc agg acc tta tta gca aaa tct gtg ttt aac 959 Ser Ile Gly Lys Val Cys Arg Thr Leu Leu Ala Lys Ser Val Phe Asn 305 310 315 agt ttg tat gca tct cca caa ctg gaa ggg ttt tca gct gaa tct agg 1007 Ser Leu Tyr Ala Ser Pro Gln Leu Glu Gly Phe Ser Ala Glu Ser Arg 320 325 330 335 aaa tta ctt ctc att gtt cag gcc ctt agg gat aac ctg gaa cct gga 1055 Lys Leu Leu Leu Ile Val Gln Ala Leu Arg Asp Asn Leu Glu Pro Gly 340 345 350 acc ttt gat att ggg ggg tta tat gaa tca att gag gag tgc ctg att 1103 Thr Phe Asp Ile Gly Gly Leu Tyr Glu Ser Ile Glu Glu Cys Leu Ile 355 360 365 aat gat ccc tgg gtt ttg ctc aat gca tct tgg ttc aac tcc ttc ctt 1151 Asn Asp Pro Trp Val Leu Leu Asn Ala Ser Trp Phe Asn Ser Phe Leu 370 375 380 aca cat gca ctg aag tagttgtagc aatgctacta tttgctatcc atactgtcca 1206 Thr His Ala Leu Lys 385 aaaaagtact cgagccccca ag 1228 77 388 PRT Equine influenza virus H3N8 77 Gly Gly Tyr Pro Asn Tyr Leu Gln Ala Trp Lys Gln Val Leu Ala Glu 1 5 10 15 Leu Gln Asp Leu Glu Asn Glu Glu Lys Thr Pro Lys Thr Lys Asn Met 20 25 30 Lys Lys Thr Ser Gln Leu Lys Trp Ala Leu Gly Glu Asn Met Ala Pro 35 40 45 Glu Lys Val Asp Phe Glu Asp Cys Lys Asp Ile Asn Asp Leu Lys Gln 50 55 60 Tyr Asp Ser Asp Glu Pro Glu Thr Arg Ser Leu Ala Ser Trp Ile Gln 65 70 75 80 Ser Glu Phe Asn Lys Ala Cys Glu Leu Thr Asp Ser Ser Trp Ile Glu 85 90 95 Leu Asp Glu Ile Gly Glu Asp Ile Ala Pro Ile Glu Tyr Ile Ala Ser 100 105 110 Met Arg Arg Asn Tyr Phe Thr Ala Glu Val Ser His Cys Arg Ala Thr 115 120 125 Glu Tyr Ile Met Lys Gly Val Tyr Ile Asn Thr Ala Leu Leu Asn Ala 130 135 140 Ser Cys Ala Ala Met Asp Glu Phe Gln Leu Ile Pro Met Ile Ser Lys 145 150 155 160 Cys Arg Thr Lys Glu Gly Arg Arg Lys Thr Asn Leu Tyr Gly Phe Ile 165 170 175 Ile Lys Gly Arg Ser His Leu Arg Asn Asp Thr Asp Val Val Asn Phe 180 185 190 Val Ser Met Glu Phe Ser Leu Thr Asp Pro Arg Phe Glu Pro His Lys 195 200 205 Trp Glu Lys Tyr Cys Val Leu Glu Ile Gly Asp Met Leu Leu Arg Thr 210 215 220 Ala Val Gly Gln Val Ser Arg Pro Met Phe Leu Tyr Val Arg Thr Asn 225 230 235 240 Gly Thr Ser Lys Ile Lys Met Lys Arg Gly Met Glu Met Arg Arg Cys 245 250 255 Leu Leu Gln Ser Leu Gln Gln Ile Glu Ser Met Ile Glu Ala Glu Ser 260 265 270 Ser Val Lys Glu Lys Asp Met Thr Lys Glu Phe Phe Glu Asn Lys Ser 275 280 285 Glu Thr Trp Pro Ile Gly Glu Ser Pro Lys Gly Val Glu Glu Gly Ser 290 295 300 Ile Gly Lys Val Cys Arg Thr Leu Leu Ala Lys Ser Val Phe Asn Ser 305 310 315 320 Leu Tyr Ala Ser Pro Gln Leu Glu Gly Phe Ser Ala Glu Ser Arg Lys 325 330 335 Leu Leu Leu Ile Val Gln Ala Leu Arg Asp Asn Leu Glu Pro Gly Thr 340 345 350 Phe Asp Ile Gly Gly Leu Tyr Glu Ser Ile Glu Glu Cys Leu Ile Asn 355 360 365 Asp Pro Trp Val Leu Leu Asn Ala Ser Trp Phe Asn Ser Phe Leu Thr 370 375 380 His Ala Leu Lys 385 78 1164 DNA Equine influenza virus H3N8 78 ggcgggtacc caaactatct ccaagcttgg aagcaagtat tagcagaact acaagacctt 60 gagaacgaag aaaagacccc taagaccaag aatatgaaaa aaacaagcca attgaaatgg 120 gcactcggtg aaaatatggc accagagaaa gtggattttg aggattgtaa agacatcaat 180 gatttgaaac agtatgacag tgatgagcca gaaacaaggt ctcttgcaag ttggattcaa 240 agtgagttca acaaagcttg tgagctgaca gattcaagct ggatagagct cgatgaaatt 300 ggggaggata ttgccccaat agaatacatt gcgagcatga ggagaaatta ttttactgct 360 gaggtttccc attgtagagc aacagaatat ataatgaagg gagtgtacat caacactgct 420 ctactcaatg catcctgtgc tgcgatggat gaattccaat taattccgat gataagcaaa 480 tgcaggacca aagaagggag aaggaagaca aatttatatg gattcataat aaagggaagg 540 tcccatttaa ggaatgatac cgacgtggta aactttgtaa gtatggaatt ttctctcact 600 gatccaagat ttgagccaca taaatgggaa aaatactgcg ttctagaaat tggagacatg 660 ctcctaagga ctgctgtagg tcaagtgtca agacccatgt ttttgtatgt aaggacaaat 720 ggaacctcta aaattaaaat gaaacgggga atggaaatga gacgctgcct ccttcagtct 780 ctgcaacaga ttgaaagcat gatcgaagct gagtcctcag tcaaagaaaa ggacatgacc 840 aaagaattct ttgagaacaa atcagagaca tggcctatag gagagtcccc caaaggagtg 900 gaagagggct caatcgggaa ggtttgcagg accttattag caaaatctgt gtttaacagt 960 ttgtatgcat ctccacaact ggaagggttt tcagctgaat ctaggaaatt acttctcatt 1020 gttcaggccc ttagggataa cctggaacct ggaacctttg atattggggg gttatatgaa 1080 tcaattgagg agtgcctgat taatgatccc tgggttttgc tcaatgcatc ttggttcaac 1140 tccttcctta cacatgcact gaag 1164 79 1223 DNA Equine influenza virus H3N8 79 ggggcgggta cccaaactat ctccaagctt ggaagcaagt attagcagaa ctacaagacc 60 ttgagaacga agaaaagacc cctaagacca agaatatgaa aaaaacaagc caattgaaat 120 gggcactcgg tgaaaatatg gcaccagaga aagtggattt tgaggattgt aaagacatca 180 atgatttgaa acagtatgac agtgatgagc cagaaacaag gtctcttgca agttggattc 240 aaagtgagtt caacaaagct tgtgagctga cagattcaag ctggatagag ctcgatgaaa 300 ttggggagga tattgcccca atagaataca ttgcgagcat gaggagaaat tattttactg 360 ctgaggtttc ccattgtaga gcaacagaat atataatgaa gggagtgtac atcaacactg 420 ctctactcaa tgcatcctgt gctgcgatgg atgaattcca attaattccg atgataagca 480 aatgcaggac caaagaaggg agaaggaaga caaatttata tggattcata ataaagggaa 540 ggtcccattt aaggaatgat accgacgtgg taaactttgt aagtatggaa ttttctctca 600 ctgatccaag atttgagcca cataaatggg aaaaatactg cgttctagaa attggagaca 660 tgctcctaag gactgctgta ggtcaagtgt caagacccat gtttttgtat gtaaggacaa 720 atggaacctc taaaattaaa atgaaatggg gaatggaaat gagacgctgc ctccttcagt 780 ctctgcaaca gattgaaagc atgatcgaag ctgagtcctc agtcaaagaa aaggacatga 840 ccaaagaatt ctttgagaac aaatcagaga catggcctat aggagagtcc cccaaaggag 900 tggaagaggg ctcaatcggg aaggtttgca ggaccttatt agcaaaatct gtgtttaaca 960 gtttgtatgc atctccacaa ctggaagggt tttcagctga atctaggaaa ttacttctca 1020 ttgttcaggc ccttagggat aacctggaac ctggaacctt tgatattggg gggttatatg 1080 aatcaattga ggagtgcctg attaatgatc cctgggtttt gctcaatgca tcttggttca 1140 actccttcct tacacatgca ctgaagtagt tgtagcaatg ctactatttg ctatccatac 1200 tgtccaaaaa agtactcgag ccc 1223 80 1233 DNA Equine influenza virus H3N8 CDS (3)..(1172) 80 at gaa aag ggt ata aac cca aac tat ctc caa gct tgg aag caa gta 47 Glu Lys Gly Ile Asn Pro Asn Tyr Leu Gln Ala Trp Lys Gln Val 1 5 10 15 tta gca gaa cta caa gac ctt gag aac gaa gaa aag acc cct aag acc 95 Leu Ala Glu Leu Gln Asp Leu Glu Asn Glu Glu Lys Thr Pro Lys Thr 20 25 30 aag aat atg aaa aaa aca agc caa ttg aaa tgg gca ctc ggt gaa aat 143 Lys Asn Met Lys Lys Thr Ser Gln Leu Lys Trp Ala Leu Gly Glu Asn 35 40 45 atg gca cca gag aaa gtg gat ttt gag gat tgt aaa gac atc aat gat 191 Met Ala Pro Glu Lys Val Asp Phe Glu Asp Cys Lys Asp Ile Asn Asp 50 55 60 ttg aaa cag tat gac agt gat gag cca gaa aca agg tct ctt gca agt 239 Leu Lys Gln Tyr Asp Ser Asp Glu Pro Glu Thr Arg Ser Leu Ala Ser 65 70 75 tgg att caa agt gag ttc aac aaa gct tgt gag ctg aca gat tca agc 287 Trp Ile Gln Ser Glu Phe Asn Lys Ala Cys Glu Leu Thr Asp Ser Ser 80 85 90 95 tgg ata gag ctc gat gaa att ggg gag gat att gcc cca ata gaa tac 335 Trp Ile Glu Leu Asp Glu Ile Gly Glu Asp Ile Ala Pro Ile Glu Tyr 100 105 110 att gcg agc atg agg aga aat tat ttt act gct gag gtt tcc cat tgt 383 Ile Ala Ser Met Arg Arg Asn Tyr Phe Thr Ala Glu Val Ser His Cys 115 120 125 aga gca aca gaa tat ata atg aag gga gtg tac atc aac act gct cta 431 Arg Ala Thr Glu Tyr Ile Met Lys Gly Val Tyr Ile Asn Thr Ala Leu 130 135 140 ctc aat gca tcc tgt gct gcg atg gat gaa ttc caa tta att ccg atg 479 Leu Asn Ala Ser Cys Ala Ala Met Asp Glu Phe Gln Leu Ile Pro Met 145 150 155 ata agc aaa tgc agg acc aaa gaa ggg aga agg aag aca aat tta tat 527 Ile Ser Lys Cys Arg Thr Lys Glu Gly Arg Arg Lys Thr Asn Leu Tyr 160 165 170 175 gga ttc ata ata aag gga agg tcc cat tta agg aat gat acc gac gtg 575 Gly Phe Ile Ile Lys Gly Arg Ser His Leu Arg Asn Asp Thr Asp Val 180 185 190 gta aac ttt gta agt atg gaa ttt tct ctc act gat cca aga ttt gag 623 Val Asn Phe Val Ser Met Glu Phe Ser Leu Thr Asp Pro Arg Phe Glu 195 200 205 cca cat aaa tgg gaa aaa tac tgc gtt cta gaa att gga gac atg ctc 671 Pro His Lys Trp Glu Lys Tyr Cys Val Leu Glu Ile Gly Asp Met Leu 210 215 220 cta agg act gct gta ggt caa gtg tca aga ccc atg ttt ttg tat gta 719 Leu Arg Thr Ala Val Gly Gln Val Ser Arg Pro Met Phe Leu Tyr Val 225 230 235 agg aca aat gga acc tct aaa att aaa atg aaa tgg gga atg gaa atg 767 Arg Thr Asn Gly Thr Ser Lys Ile Lys Met Lys Trp Gly Met Glu Met 240 245 250 255 aga cgc tgc ctc ctt cag tct ctg caa cag att gaa agc atg atc gaa 815 Arg Arg Cys Leu Leu Gln Ser Leu Gln Gln Ile Glu Ser Met Ile Glu 260 265 270 gct gag tcc tca gtc aaa gaa aag gac atg acc aaa gaa ttc ttt gag 863 Ala Glu Ser Ser Val Lys Glu Lys Asp Met Thr Lys Glu Phe Phe Glu 275 280 285 aac aaa tca gag aca tgg cct ata gga gag tcc ccc aaa gga gtg gaa 911 Asn Lys Ser Glu Thr Trp Pro Ile Gly Glu Ser Pro Lys Gly Val Glu 290 295 300 gag ggc tca atc ggg aag gtt tgc agg acc tta tta gca aaa tct gtg 959 Glu Gly Ser Ile Gly Lys Val Cys Arg Thr Leu Leu Ala Lys Ser Val 305 310 315 ttt aac agt ttg tat gca tct cca caa ctg gaa ggg ttt tca gct gaa 1007 Phe Asn Ser Leu Tyr Ala Ser Pro Gln Leu Glu Gly Phe Ser Ala Glu 320 325 330 335 tct agg aaa tta ctt ctc att gtt cag gcc ctt agg gat aac ctg gaa 1055 Ser Arg Lys Leu Leu Leu Ile Val Gln Ala Leu Arg Asp Asn Leu Glu 340 345 350 cct gga acc ttt gat att ggg ggg tta tat gaa tca att gag gag tgc 1103 Pro Gly Thr Phe Asp Ile Gly Gly Leu Tyr Glu Ser Ile Glu Glu Cys 355 360 365 ctg att aat gat ccc tgg gtt ttg ctc aat gca tct tgg ttc aac tcc 1151 Leu Ile Asn Asp Pro Trp Val Leu Leu Asn Ala Ser Trp Phe Asn Ser 370 375 380 ttc ctt aca cat gca ctg aag tagttgtagc aatgctacta tttgctatcc 1202 Phe Leu Thr His Ala Leu Lys 385 390 atactgtcca aaaaagtacc ttgtttctac t 1233 81 390 PRT Equine influenza virus H3N8 81 Glu Lys Gly Ile Asn Pro Asn Tyr Leu Gln Ala Trp Lys Gln Val Leu 1 5 10 15 Ala Glu Leu Gln Asp Leu Glu Asn Glu Glu Lys Thr Pro Lys Thr Lys 20 25 30 Asn Met Lys Lys Thr Ser Gln Leu Lys Trp Ala Leu Gly Glu Asn Met 35 40 45 Ala Pro Glu Lys Val Asp Phe Glu Asp Cys Lys Asp Ile Asn Asp Leu 50 55 60 Lys Gln Tyr Asp Ser Asp Glu Pro Glu Thr Arg Ser Leu Ala Ser Trp 65 70 75 80 Ile Gln Ser Glu Phe Asn Lys Ala Cys Glu Leu Thr Asp Ser Ser Trp 85 90 95 Ile Glu Leu Asp Glu Ile Gly Glu Asp Ile Ala Pro Ile Glu Tyr Ile 100 105 110 Ala Ser Met Arg Arg Asn Tyr Phe Thr Ala Glu Val Ser His Cys Arg 115 120 125 Ala Thr Glu Tyr Ile Met Lys Gly Val Tyr Ile Asn Thr Ala Leu Leu 130 135 140 Asn Ala Ser Cys Ala Ala Met Asp Glu Phe Gln Leu Ile Pro Met Ile 145 150 155 160 Ser Lys Cys Arg Thr Lys Glu Gly Arg Arg Lys Thr Asn Leu Tyr Gly 165 170 175 Phe Ile Ile Lys Gly Arg Ser His Leu Arg Asn Asp Thr Asp Val Val 180 185 190 Asn Phe Val Ser Met Glu Phe Ser Leu Thr Asp Pro Arg Phe Glu Pro 195 200 205 His Lys Trp Glu Lys Tyr Cys Val Leu Glu Ile Gly Asp Met Leu Leu 210 215 220 Arg Thr Ala Val Gly Gln Val Ser Arg Pro Met Phe Leu Tyr Val Arg 225 230 235 240 Thr Asn Gly Thr Ser Lys Ile Lys Met Lys Trp Gly Met Glu Met Arg 245 250 255 Arg Cys Leu Leu Gln Ser Leu Gln Gln Ile Glu Ser Met Ile Glu Ala 260 265 270 Glu Ser Ser Val Lys Glu Lys Asp Met Thr Lys Glu Phe Phe Glu Asn 275 280 285 Lys Ser Glu Thr Trp Pro Ile Gly Glu Ser Pro Lys Gly Val Glu Glu 290 295 300 Gly Ser Ile Gly Lys Val Cys Arg Thr Leu Leu Ala Lys Ser Val Phe 305 310 315 320 Asn Ser Leu Tyr Ala Ser Pro Gln Leu Glu Gly Phe Ser Ala Glu Ser 325 330 335 Arg Lys Leu Leu Leu Ile Val Gln Ala Leu Arg Asp Asn Leu Glu Pro 340 345 350 Gly Thr Phe Asp Ile Gly Gly Leu Tyr Glu Ser Ile Glu Glu Cys Leu 355 360 365 Ile Asn Asp Pro Trp Val Leu Leu Asn Ala Ser Trp Phe Asn Ser Phe 370 375 380 Leu Thr His Ala Leu Lys 385 390 82 1170 DNA Equine influenza virus H3N8 82 gaaaagggta taaacccaaa ctatctccaa gcttggaagc aagtattagc agaactacaa 60 gaccttgaga acgaagaaaa gacccctaag accaagaata tgaaaaaaac aagccaattg 120 aaatgggcac tcggtgaaaa tatggcacca gagaaagtgg attttgagga ttgtaaagac 180 atcaatgatt tgaaacagta tgacagtgat gagccagaaa caaggtctct tgcaagttgg 240 attcaaagtg agttcaacaa agcttgtgag ctgacagatt caagctggat agagctcgat 300 gaaattgggg aggatattgc cccaatagaa tacattgcga gcatgaggag aaattatttt 360 actgctgagg tttcccattg tagagcaaca gaatatataa tgaagggagt gtacatcaac 420 actgctctac tcaatgcatc ctgtgctgcg atggatgaat tccaattaat tccgatgata 480 agcaaatgca ggaccaaaga agggagaagg aagacaaatt tatatggatt cataataaag 540 ggaaggtccc atttaaggaa tgataccgac gtggtaaact ttgtaagtat ggaattttct 600 ctcactgatc caagatttga gccacataaa tgggaaaaat actgcgttct agaaattgga 660 gacatgctcc taaggactgc tgtaggtcaa gtgtcaagac ccatgttttt gtatgtaagg 720 acaaatggaa cctctaaaat taaaatgaaa tggggaatgg aaatgagacg ctgcctcctt 780 cagtctctgc aacagattga aagcatgatc gaagctgagt cctcagtcaa agaaaaggac 840 atgaccaaag aattctttga gaacaaatca gagacatggc ctataggaga gtcccccaaa 900 ggagtggaag agggctcaat cgggaaggtt tgcaggacct tattagcaaa atctgtgttt 960 aacagtttgt atgcatctcc acaactggaa gggttttcag ctgaatctag gaaattactt 1020 ctcattgttc aggcccttag ggataacctg gaacctggaa cctttgatat tggggggtta 1080 tatgaatcaa ttgaggagtg cctgattaat gatccctggg ttttgctcaa tgcatcttgg 1140 ttcaactcct tccttacaca tgcactgaag 1170 83 25 DNA Artificial sequence Synthetic Primer 83 ggggcgggta cccaaactat ctcca 25 84 27 DNA Artificial sequence Synthetic Primer 84 gggggctcga gtactttttt ggacagt 27 85 1234 DNA Equine influenza virus H3N8 CDS (1)..(1188) 85 atg aaa ttg aga act caa ata cca gca gaa atg ctc gca agc att gat 48 Met Lys Leu Arg Thr Gln Ile Pro Ala Glu Met Leu Ala Ser Ile Asp 1 5 10 15 ctg aaa tat ttc aat gat tca aca aaa aag aaa att gag aag ata cga 96 Leu Lys Tyr Phe Asn Asp Ser Thr Lys Lys Lys Ile Glu Lys Ile Arg 20 25 30 cca ctt ctg gtc gat ggg act gct tca ctg agt cct ggc atg atg atg 144 Pro Leu Leu Val Asp Gly Thr Ala Ser Leu Ser Pro Gly Met Met Met 35 40 45 gga atg ttc aac atg ttg agc act gta cta ggt gta tcc ata tta aac 192 Gly Met Phe Asn Met Leu Ser Thr Val Leu Gly Val Ser Ile Leu Asn 50 55 60 ctg ggc cag agg aaa tac aca aag acc aca tac tgg tgg gat ggt ctg 240 Leu Gly Gln Arg Lys Tyr Thr Lys Thr Thr Tyr Trp Trp Asp Gly Leu 65 70 75 80 caa tca tcc gat gat ttt gct ttg ata gtg aat gcg cct aat cat gaa 288 Gln Ser Ser Asp Asp Phe Ala Leu Ile Val Asn Ala Pro Asn His Glu 85 90 95 gga ata cag gct gga gta gac aga ttc tat aga act tgc aaa ctg gtc 336 Gly Ile Gln Ala Gly Val Asp Arg Phe Tyr Arg Thr Cys Lys Leu Val 100 105 110 ggg atc aac atg agc aaa aag aag tcc tac ata aat aga acc ggc aca 384 Gly Ile Asn Met Ser Lys Lys Lys Ser Tyr Ile Asn Arg Thr Gly Thr 115 120 125 ttc gaa ttc aca agc ttt ttc tac cgg tat ggt ttt gtc gcc aat ttc 432 Phe Glu Phe Thr Ser Phe Phe Tyr Arg Tyr Gly Phe Val Ala Asn Phe 130 135 140 agc atg gag cta ccc agt ttt ggg gtt tcc ggg ata aat gaa tct gca 480 Ser Met Glu Leu Pro Ser Phe Gly Val Ser Gly Ile Asn Glu Ser Ala 145 150 155 160 gac atg agc att gga atg aca gtt atc aaa aac aac atg ata aat aat 528 Asp Met Ser Ile Gly Met Thr Val Ile Lys Asn Asn Met Ile Asn Asn 165 170 175 gat ctc ggt ccc gcc acg gca caa atg gca ctc caa ctc ttc att aag 576 Asp Leu Gly Pro Ala Thr Ala Gln Met Ala Leu Gln Leu Phe Ile Lys 180 185 190 gat tat cgg tac aca tac cgg tgc cat aga ggc gat acc cag ata caa 624 Asp Tyr Arg Tyr Thr Tyr Arg Cys His Arg Gly Asp Thr Gln Ile Gln 195 200 205 acc aga aga tcc ttt gag ttg aag aaa ctg tgg gaa cag act cga tca 672 Thr Arg Arg Ser Phe Glu Leu Lys Lys Leu Trp Glu Gln Thr Arg Ser 210 215 220 aag act ggt cta ctg gta tca gat ggg ggt cca aac cta tac aac atc 720 Lys Thr Gly Leu Leu Val Ser Asp Gly Gly Pro Asn Leu Tyr Asn Ile 225 230 235 240 aga aac cta cac atc ccg gaa gtc tgt ttg aaa tgg gag ctg atg gat 768 Arg Asn Leu His Ile Pro Glu Val Cys Leu Lys Trp Glu Leu Met Asp 245 250 255 gaa gat tat aaa ggg agg cta tgt aat cca ttg aat cct ttc gtt agc 816 Glu Asp Tyr Lys Gly Arg Leu Cys Asn Pro Leu Asn Pro Phe Val Ser 260 265 270 cac aaa gaa att gaa tca gtg aac agt gca gta gta atg cct gcg cat 864 His Lys Glu Ile Glu Ser Val Asn Ser Ala Val Val Met Pro Ala His 275 280 285 ggc cct gcc aaa agc atg gag tat gat gct gtt gca aca aca cac tct 912 Gly Pro Ala Lys Ser Met Glu Tyr Asp Ala Val Ala Thr Thr His Ser 290 295 300 tgg atc ccc aag agg aac cgg tcc ata ttg aac aca agt caa agg gga 960 Trp Ile Pro Lys Arg Asn Arg Ser Ile Leu Asn Thr Ser Gln Arg Gly 305 310 315 320 ata ctc gaa gat gag cag atg tat cag aaa tgc tgc aac ctg ttt gaa 1008 Ile Leu Glu Asp Glu Gln Met Tyr Gln Lys Cys Cys Asn Leu Phe Glu 325 330 335 aaa ttc ttc ccc agc agc tca tac aga aga cca gtc gga att tct agt 1056 Lys Phe Phe Pro Ser Ser Ser Tyr Arg Arg Pro Val Gly Ile Ser Ser 340 345 350 atg gtt gag gcc atg gtg tcc agg gcc cgc att gat gca cga att gac 1104 Met Val Glu Ala Met Val Ser Arg Ala Arg Ile Asp Ala Arg Ile Asp 355 360 365 ttc gaa tct gga cgg ata aag aag gat gag ttc gct gag atc atg aag 1152 Phe Glu Ser Gly Arg Ile Lys Lys Asp Glu Phe Ala Glu Ile Met Lys 370 375 380 atc tgt tcc acc att gaa gag ctc aga cgg caa aaa tagtgaattt 1198 Ile Cys Ser Thr Ile Glu Glu Leu Arg Arg Gln Lys 385 390 395 agcttgatct tcgtgaaaaa atgccttgtt tctact 1234 86 396 PRT Equine influenza virus H3N8 86 Met Lys Leu Arg Thr Gln Ile Pro Ala Glu Met Leu Ala Ser Ile Asp 1 5 10 15 Leu Lys Tyr Phe Asn Asp Ser Thr Lys Lys Lys Ile Glu Lys Ile Arg 20 25 30 Pro Leu Leu Val Asp Gly Thr Ala Ser Leu Ser Pro Gly Met Met Met 35 40 45 Gly Met Phe Asn Met Leu Ser Thr Val Leu Gly Val Ser Ile Leu Asn 50 55 60 Leu Gly Gln Arg Lys Tyr Thr Lys Thr Thr Tyr Trp Trp Asp Gly Leu 65 70 75 80 Gln Ser Ser Asp Asp Phe Ala Leu Ile Val Asn Ala Pro Asn His Glu 85 90 95 Gly Ile Gln Ala Gly Val Asp Arg Phe Tyr Arg Thr Cys Lys Leu Val 100 105 110 Gly Ile Asn Met Ser Lys Lys Lys Ser Tyr Ile Asn Arg Thr Gly Thr 115 120 125 Phe Glu Phe Thr Ser Phe Phe Tyr Arg Tyr Gly Phe Val Ala Asn Phe 130 135 140 Ser Met Glu Leu Pro Ser Phe Gly Val Ser Gly Ile Asn Glu Ser Ala 145 150 155 160 Asp Met Ser Ile Gly Met Thr Val Ile Lys Asn Asn Met Ile Asn Asn 165 170 175 Asp Leu Gly Pro Ala Thr Ala Gln Met Ala Leu Gln Leu Phe Ile Lys 180 185 190 Asp Tyr Arg Tyr Thr Tyr Arg Cys His Arg Gly Asp Thr Gln Ile Gln 195 200 205 Thr Arg Arg Ser Phe Glu Leu Lys Lys Leu Trp Glu Gln Thr Arg Ser 210 215 220 Lys Thr Gly Leu Leu Val Ser Asp Gly Gly Pro Asn Leu Tyr Asn Ile 225 230 235 240 Arg Asn Leu His Ile Pro Glu Val Cys Leu Lys Trp Glu Leu Met Asp 245 250 255 Glu Asp Tyr Lys Gly Arg Leu Cys Asn Pro Leu Asn Pro Phe Val Ser 260 265 270 His Lys Glu Ile Glu Ser Val Asn Ser Ala Val Val Met Pro Ala His 275 280 285 Gly Pro Ala Lys Ser Met Glu Tyr Asp Ala Val Ala Thr Thr His Ser 290 295 300 Trp Ile Pro Lys Arg Asn Arg Ser Ile Leu Asn Thr Ser Gln Arg Gly 305 310 315 320 Ile Leu Glu Asp Glu Gln Met Tyr Gln Lys Cys Cys Asn Leu Phe Glu 325 330 335 Lys Phe Phe Pro Ser Ser Ser Tyr Arg Arg Pro Val Gly Ile Ser Ser 340 345 350 Met Val Glu Ala Met Val Ser Arg Ala Arg Ile Asp Ala Arg Ile Asp 355 360 365 Phe Glu Ser Gly Arg Ile Lys Lys Asp Glu Phe Ala Glu Ile Met Lys 370 375 380 Ile Cys Ser Thr Ile Glu Glu Leu Arg Arg Gln Lys 385 390 395 87 1188 DNA Equine influenza virus H3N8 87 atgaaattga gaactcaaat accagcagaa atgctcgcaa gcattgatct gaaatatttc 60 aatgattcaa caaaaaagaa aattgagaag atacgaccac ttctggtcga tgggactgct 120 tcactgagtc ctggcatgat gatgggaatg ttcaacatgt tgagcactgt actaggtgta 180 tccatattaa acctgggcca gaggaaatac acaaagacca catactggtg ggatggtctg 240 caatcatccg atgattttgc tttgatagtg aatgcgccta atcatgaagg aatacaggct 300 ggagtagaca gattctatag aacttgcaaa ctggtcggga tcaacatgag caaaaagaag 360 tcctacataa atagaaccgg cacattcgaa ttcacaagct ttttctaccg gtatggtttt 420 gtcgccaatt tcagcatgga gctacccagt tttggggttt ccgggataaa tgaatctgca 480 gacatgagca ttggaatgac agttatcaaa aacaacatga taaataatga tctcggtccc 540 gccacggcac aaatggcact ccaactcttc attaaggatt atcggtacac ataccggtgc 600 catagaggcg atacccagat acaaaccaga agatcctttg agttgaagaa actgtgggaa 660 cagactcgat caaagactgg tctactggta tcagatgggg gtccaaacct atacaacatc 720 agaaacctac acatcccgga agtctgtttg aaatgggagc tgatggatga agattataaa 780 gggaggctat gtaatccatt gaatcctttc gttagccaca aagaaattga atcagtgaac 840 agtgcagtag taatgcctgc gcatggccct gccaaaagca tggagtatga tgctgttgca 900 acaacacact cttggatccc caagaggaac cggtccatat tgaacacaag tcaaagggga 960 atactcgaag atgagcagat gtatcagaaa tgctgcaacc tgtttgaaaa attcttcccc 1020 agcagctcat acagaagacc agtcggaatt tctagtatgg ttgaggccat ggtgtccagg 1080 gcccgcattg atgcacgaat tgacttcgaa tctggacgga taaagaagga tgagttcgct 1140 gagatcatga agatctgttc caccattgaa gagctcagac ggcaaaaa 1188 88 1240 DNA Equine influenza virus H3N8 CDS (8)..(1195) 88 caaaagt atg aaa ttg aga act caa ata cca gca gaa atg ctc gca agc 49 Met Lys Leu Arg Thr Gln Ile Pro Ala Glu Met Leu Ala Ser 1 5 10 att gat ctg aaa tat ttc aat gat tca aca aaa aag aaa att gag aag 97 Ile Asp Leu Lys Tyr Phe Asn Asp Ser Thr Lys Lys Lys Ile Glu Lys 15 20 25 30 ata cga cca ctt ctg gtc gat ggg act gct tca ctg agt cct ggc atg 145 Ile Arg Pro Leu Leu Val Asp Gly Thr Ala Ser Leu Ser Pro Gly Met 35 40 45 atg atg gga atg ttc aac atg ttg agc act gta cta ggt gta tcc ata 193 Met Met Gly Met Phe Asn Met Leu Ser Thr Val Leu Gly Val Ser Ile 50 55 60 tta aac ctg ggc cag agg aaa tac aca aag acc aca tac tgg tgg gat 241 Leu Asn Leu Gly Gln Arg Lys Tyr Thr Lys Thr Thr Tyr Trp Trp Asp 65 70 75 ggt ctg caa tca tcc gat gat ttt gct ttg ata gtg aat gcg cct aat 289 Gly Leu Gln Ser Ser Asp Asp Phe Ala Leu Ile Val Asn Ala Pro Asn 80 85 90 cat gaa gga ata cag gct gga gta gac aga ttc tat aga act tgc aaa 337 His Glu Gly Ile Gln Ala Gly Val Asp Arg Phe Tyr Arg Thr Cys Lys 95 100 105 110 ctg gtc ggg atc aac atg agc aaa aag aag tcc tac ata aat aga acc 385 Leu Val Gly Ile Asn Met Ser Lys Lys Lys Ser Tyr Ile Asn Arg Thr 115 120 125 ggc tca ttc gaa ttc aca agc ttt ttc tac cgg tat ggt ttt gtc gcc 433 Gly Ser Phe Glu Phe Thr Ser Phe Phe Tyr Arg Tyr Gly Phe Val Ala 130 135 140 aat ttc agc atg gag cta ccc agt ttt ggg gtt tcc ggg ata aat gaa 481 Asn Phe Ser Met Glu Leu Pro Ser Phe Gly Val Ser Gly Ile Asn Glu 145 150 155 tct gca gac atg agc att gga atg aca gtt atc aaa aac aac atg ata 529 Ser Ala Asp Met Ser Ile Gly Met Thr Val Ile Lys Asn Asn Met Ile 160 165 170 aat aat gat ctc ggt ccc gcc acg gca caa atg gca ctc caa ctc ttc 577 Asn Asn Asp Leu Gly Pro Ala Thr Ala Gln Met Ala Leu Gln Leu Phe 175 180 185 190 att aag gat tat cgg tac aca tac cgg tgc cat aga ggc gat acc cag 625 Ile Lys Asp Tyr Arg Tyr Thr Tyr Arg Cys His Arg Gly Asp Thr Gln 195 200 205 ata caa acc aga aga tcc ttt gag ttg aag aaa ctg tgg gaa cag act 673 Ile Gln Thr Arg Arg Ser Phe Glu Leu Lys Lys Leu Trp Glu Gln Thr 210 215 220 cga tca aag act ggt cta ctg gta tca gat ggg ggt cca aac cta tac 721 Arg Ser Lys Thr Gly Leu Leu Val Ser Asp Gly Gly Pro Asn Leu Tyr 225 230 235 aac atc aga aac cta cac atc ccg gaa gtc tgt ttg aaa tgg gag ctg 769 Asn Ile Arg Asn Leu His Ile Pro Glu Val Cys Leu Lys Trp Glu Leu 240 245 250 atg gat gaa gat tat aaa ggg agg cta tgt aat cca ttg aat cct ttc 817 Met Asp Glu Asp Tyr Lys Gly Arg Leu Cys Asn Pro Leu Asn Pro Phe 255 260 265 270 gtt agc cac aaa gaa att gaa tca gtg aac agt gca gta gta atg cct 865 Val Ser His Lys Glu Ile Glu Ser Val Asn Ser Ala Val Val Met Pro 275 280 285 gcg cat ggc cct gcc aaa agc atg gag tat gat gct gtt gca aca aca 913 Ala His Gly Pro Ala Lys Ser Met Glu Tyr Asp Ala Val Ala Thr Thr 290 295 300 cac tct tgg atc ccc aag agg aac cgg tcc ata ttg aac aca agt caa 961 His Ser Trp Ile Pro Lys Arg Asn Arg Ser Ile Leu Asn Thr Ser Gln 305 310 315 agg gga ata ctc gaa gat gag cag atg tat cag aaa tgc tgc aac ctg 1009 Arg Gly Ile Leu Glu Asp Glu Gln Met Tyr Gln Lys Cys Cys Asn Leu 320 325 330 ttt gaa aaa ttc ttc ccc agc agc tca tac aga aga cca gtc gga att 1057 Phe Glu Lys Phe Phe Pro Ser Ser Ser Tyr Arg Arg Pro Val Gly Ile 335 340 345 350 tct agt atg gtt gag gcc atg gtg tcc agg gcc cgc att gat gca cga 1105 Ser Ser Met Val Glu Ala Met Val Ser Arg Ala Arg Ile Asp Ala Arg 355 360 365 att gac ttc gaa tct gga cgg ata aag aag gat gag ttc gct gag atc 1153 Ile Asp Phe Glu Ser Gly Arg Ile Lys Lys Asp Glu Phe Ala Glu Ile 370 375 380 atg aag atc tgt tcc acc att gaa gag ctc aga cgg caa aaa 1195 Met Lys Ile Cys Ser Thr Ile Glu Glu Leu Arg Arg Gln Lys 385 390 395 tagtgaattt agcttgatct tcgtgaaaaa atgccttgtt ctact 1240 89 396 PRT Equine influenza virus H3N8 89 Met Lys Leu Arg Thr Gln Ile Pro Ala Glu Met Leu Ala Ser Ile Asp 1 5 10 15 Leu Lys Tyr Phe Asn Asp Ser Thr Lys Lys Lys Ile Glu Lys Ile Arg 20 25 30 Pro Leu Leu Val Asp Gly Thr Ala Ser Leu Ser Pro Gly Met Met Met 35 40 45 Gly Met Phe Asn Met Leu Ser Thr Val Leu Gly Val Ser Ile Leu Asn 50 55 60 Leu Gly Gln Arg Lys Tyr Thr Lys Thr Thr Tyr Trp Trp Asp Gly Leu 65 70 75 80 Gln Ser Ser Asp Asp Phe Ala Leu Ile Val Asn Ala Pro Asn His Glu 85 90 95 Gly Ile Gln Ala Gly Val Asp Arg Phe Tyr Arg Thr Cys Lys Leu Val 100 105 110 Gly Ile Asn Met Ser Lys Lys Lys Ser Tyr Ile Asn Arg Thr Gly Ser 115 120 125 Phe Glu Phe Thr Ser Phe Phe Tyr Arg Tyr Gly Phe Val Ala Asn Phe 130 135 140 Ser Met Glu Leu Pro Ser Phe Gly Val Ser Gly Ile Asn Glu Ser Ala 145 150 155 160 Asp Met Ser Ile Gly Met Thr Val Ile Lys Asn Asn Met Ile Asn Asn 165 170 175 Asp Leu Gly Pro Ala Thr Ala Gln Met Ala Leu Gln Leu Phe Ile Lys 180 185 190 Asp Tyr Arg Tyr Thr Tyr Arg Cys His Arg Gly Asp Thr Gln Ile Gln 195 200 205 Thr Arg Arg Ser Phe Glu Leu Lys Lys Leu Trp Glu Gln Thr Arg Ser 210 215 220 Lys Thr Gly Leu Leu Val Ser Asp Gly Gly Pro Asn Leu Tyr Asn Ile 225 230 235 240 Arg Asn Leu His Ile Pro Glu Val Cys Leu Lys Trp Glu Leu Met Asp 245 250 255 Glu Asp Tyr Lys Gly Arg Leu Cys Asn Pro Leu Asn Pro Phe Val Ser 260 265 270 His Lys Glu Ile Glu Ser Val Asn Ser Ala Val Val Met Pro Ala His 275 280 285 Gly Pro Ala Lys Ser Met Glu Tyr Asp Ala Val Ala Thr Thr His Ser 290 295 300 Trp Ile Pro Lys Arg Asn Arg Ser Ile Leu Asn Thr Ser Gln Arg Gly 305 310 315 320 Ile Leu Glu Asp Glu Gln Met Tyr Gln Lys Cys Cys Asn Leu Phe Glu 325 330 335 Lys Phe Phe Pro Ser Ser Ser Tyr Arg Arg Pro Val Gly Ile Ser Ser 340 345 350 Met Val Glu Ala Met Val Ser Arg Ala Arg Ile Asp Ala Arg Ile Asp 355 360 365 Phe Glu Ser Gly Arg Ile Lys Lys Asp Glu Phe Ala Glu Ile Met Lys 370 375 380 Ile Cys Ser Thr Ile Glu Glu Leu Arg Arg Gln Lys 385 390 395 90 1188 DNA Equine influenza virus H3N8 90 atgaaattga gaactcaaat accagcagaa atgctcgcaa gcattgatct gaaatatttc 60 aatgattcaa caaaaaagaa aattgagaag atacgaccac ttctggtcga tgggactgct 120 tcactgagtc ctggcatgat gatgggaatg ttcaacatgt tgagcactgt actaggtgta 180 tccatattaa acctgggcca gaggaaatac acaaagacca catactggtg ggatggtctg 240 caatcatccg atgattttgc tttgatagtg aatgcgccta atcatgaagg aatacaggct 300 ggagtagaca gattctatag aacttgcaaa ctggtcggga tcaacatgag caaaaagaag 360 tcctacataa atagaaccgg ctcattcgaa ttcacaagct ttttctaccg gtatggtttt 420 gtcgccaatt tcagcatgga gctacccagt tttggggttt ccgggataaa tgaatctgca 480 gacatgagca ttggaatgac agttatcaaa aacaacatga taaataatga tctcggtccc 540 gccacggcac aaatggcact ccaactcttc attaaggatt atcggtacac ataccggtgc 600 catagaggcg atacccagat acaaaccaga agatcctttg agttgaagaa actgtgggaa 660 cagactcgat caaagactgg tctactggta tcagatgggg gtccaaacct atacaacatc 720 agaaacctac acatcccgga agtctgtttg aaatgggagc tgatggatga agattataaa 780 gggaggctat gtaatccatt gaatcctttc gttagccaca aagaaattga atcagtgaac 840 agtgcagtag taatgcctgc gcatggccct gccaaaagca tggagtatga tgctgttgca 900 acaacacact cttggatccc caagaggaac cggtccatat tgaacacaag tcaaagggga 960 atactcgaag atgagcagat gtatcagaaa tgctgcaacc tgtttgaaaa attcttcccc 1020 agcagctcat acagaagacc agtcggaatt tctagtatgg ttgaggccat ggtgtccagg 1080 gcccgcattg atgcacgaat tgacttcgaa tctggacgga taaagaagga tgagttcgct 1140 gagatcatga agatctgttc caccattgaa gagctcagac ggcaaaaa 1188 91 1241 DNA Equine influenza virus H3N8 CDS (8)..(1195) 91 caaaagt atg aaa ttg aga act caa ata cca gca gaa atg ctc gca agc 49 Met Lys Leu Arg Thr Gln Ile Pro Ala Glu Met Leu Ala Ser 1 5 10 att gat ctg aaa tat ttc aat gat tca aca aaa aag aaa att gag aag 97 Ile Asp Leu Lys Tyr Phe Asn Asp Ser Thr Lys Lys Lys Ile Glu Lys 15 20 25 30 ata cga cca ctt ctg gtc gat ggg act gct tca ctg agt cct ggc atg 145 Ile Arg Pro Leu Leu Val Asp Gly Thr Ala Ser Leu Ser Pro Gly Met 35 40 45 atg atg gga atg ttc aac atg ttg agc act gta cta ggt gta tcc ata 193 Met Met Gly Met Phe Asn Met Leu Ser Thr Val Leu Gly Val Ser Ile 50 55 60 tta aac ctg ggc cag agg aaa tac aca aag acc aca tac tgg tgg gat 241 Leu Asn Leu Gly Gln Arg Lys Tyr Thr Lys Thr Thr Tyr Trp Trp Asp 65 70 75 ggt ctg caa tca tcc gat gat ttt gct ttg ata gtg aat gcg cct aat 289 Gly Leu Gln Ser Ser Asp Asp Phe Ala Leu Ile Val Asn Ala Pro Asn 80 85 90 cat gaa gga ata cag gct gga gta gac aga ttc tat aga act tgc aaa 337 His Glu Gly Ile Gln Ala Gly Val Asp Arg Phe Tyr Arg Thr Cys Lys 95 100 105 110 ctg gtc ggg atc aac atg agc aaa aag aag tcc tac ata aat aga acc 385 Leu Val Gly Ile Asn Met Ser Lys Lys Lys Ser Tyr Ile Asn Arg Thr 115 120 125 ggc aca ttc gaa ttc aca agc ttt ttc tac cgg tat ggt ttt gtc gcc 433 Gly Thr Phe Glu Phe Thr Ser Phe Phe Tyr Arg Tyr Gly Phe Val Ala 130 135 140 aat ttc agc atg gag cta ccc agt ttt ggg gtt tcc ggg ata aat gaa 481 Asn Phe Ser Met Glu Leu Pro Ser Phe Gly Val Ser Gly Ile Asn Glu 145 150 155 tct gca gac atg agc att gga atg aca gtt atc aaa aac aac atg ata 529 Ser Ala Asp Met Ser Ile Gly Met Thr Val Ile Lys Asn Asn Met Ile 160 165 170 aat aat gat ctc ggt ccc gcc acg gca caa atg gca ctc caa ctc ttc 577 Asn Asn Asp Leu Gly Pro Ala Thr Ala Gln Met Ala Leu Gln Leu Phe 175 180 185 190 att aag gat tat cgg tac aca tac cgg tgt caa aga ggc gat acc cag 625 Ile Lys Asp Tyr Arg Tyr Thr Tyr Arg Cys Gln Arg Gly Asp Thr Gln 195 200 205 ata caa acc aga aga tcc ttt gag ttg aag aaa ctg tgg gaa cag act 673 Ile Gln Thr Arg Arg Ser Phe Glu Leu Lys Lys Leu Trp Glu Gln Thr 210 215 220 cga tca aag act ggt cta ctg gta tca gat ggg ggt cca aac cta tac 721 Arg Ser Lys Thr Gly Leu Leu Val Ser Asp Gly Gly Pro Asn Leu Tyr 225 230 235 aac atc aga aac cta cac atc ccg gaa gtc tgt ttg aaa tgg gag ctg 769 Asn Ile Arg Asn Leu His Ile Pro Glu Val Cys Leu Lys Trp Glu Leu 240 245 250 atg gat gaa gat tat aaa ggg agg cta tgt aat cca ttg aat cct ttc 817 Met Asp Glu Asp Tyr Lys Gly Arg Leu Cys Asn Pro Leu Asn Pro Phe 255 260 265 270 gtt agc cac aaa gaa att gaa tca gtg aac agt gca gta gta atg cct 865 Val Ser His Lys Glu Ile Glu Ser Val Asn Ser Ala Val Val Met Pro 275 280 285 gcg cat ggc cct gcc aaa agc atg gag tat gat gct gtt gca aca aca 913 Ala His Gly Pro Ala Lys Ser Met Glu Tyr Asp Ala Val Ala Thr Thr 290 295 300 cac tct tgg atc ccc aag agg aac cgg tcc ata ttg aac aca agt caa 961 His Ser Trp Ile Pro Lys Arg Asn Arg Ser Ile Leu Asn Thr Ser Gln 305 310 315 agg gga ata ctc gaa gat gag cag atg tat cag aaa tgc tgc aac ctg 1009 Arg Gly Ile Leu Glu Asp Glu Gln Met Tyr Gln Lys Cys Cys Asn Leu 320 325 330 ttt gaa aaa ttc ttc ccc agc agc tca tac aga aaa cca gtc gga att 1057 Phe Glu Lys Phe Phe Pro Ser Ser Ser Tyr Arg Lys Pro Val Gly Ile 335 340 345 350 tct agt atg gtt gag gcc atg gtg tcc agg gcc cgc att gat gca cga 1105 Ser Ser Met Val Glu Ala Met Val Ser Arg Ala Arg Ile Asp Ala Arg 355 360 365 att gac ttc gaa tct gga cgg ata aag aag gat gag ttc gct gag atc 1153 Ile Asp Phe Glu Ser Gly Arg Ile Lys Lys Asp Glu Phe Ala Glu Ile 370 375 380 atg aag atc tgt tcc acc att gaa gag ctc aga cgg caa aaa 1195 Met Lys Ile Cys Ser Thr Ile Glu Glu Leu Arg Arg Gln Lys 385 390 395 tagtgaattt agcttgatct tcgtgaaaaa atgccttgtt tctact 1241 92 396 PRT Equine influenza virus H3N8 92 Met Lys Leu Arg Thr Gln Ile Pro Ala Glu Met Leu Ala Ser Ile Asp 1 5 10 15 Leu Lys Tyr Phe Asn Asp Ser Thr Lys Lys Lys Ile Glu Lys Ile Arg 20 25 30 Pro Leu Leu Val Asp Gly Thr Ala Ser Leu Ser Pro Gly Met Met Met 35 40 45 Gly Met Phe Asn Met Leu Ser Thr Val Leu Gly Val Ser Ile Leu Asn 50 55 60 Leu Gly Gln Arg Lys Tyr Thr Lys Thr Thr Tyr Trp Trp Asp Gly Leu 65 70 75 80 Gln Ser Ser Asp Asp Phe Ala Leu Ile Val Asn Ala Pro Asn His Glu 85 90 95 Gly Ile Gln Ala Gly Val Asp Arg Phe Tyr Arg Thr Cys Lys Leu Val 100 105 110 Gly Ile Asn Met Ser Lys Lys Lys Ser Tyr Ile Asn Arg Thr Gly Thr 115 120 125 Phe Glu Phe Thr Ser Phe Phe Tyr Arg Tyr Gly Phe Val Ala Asn Phe 130 135 140 Ser Met Glu Leu Pro Ser Phe Gly Val Ser Gly Ile Asn Glu Ser Ala 145 150 155 160 Asp Met Ser Ile Gly Met Thr Val Ile Lys Asn Asn Met Ile Asn Asn 165 170 175 Asp Leu Gly Pro Ala Thr Ala Gln Met Ala Leu Gln Leu Phe Ile Lys 180 185 190 Asp Tyr Arg Tyr Thr Tyr Arg Cys Gln Arg Gly Asp Thr Gln Ile Gln 195 200 205 Thr Arg Arg Ser Phe Glu Leu Lys Lys Leu Trp Glu Gln Thr Arg Ser 210 215 220 Lys Thr Gly Leu Leu Val Ser Asp Gly Gly Pro Asn Leu Tyr Asn Ile 225 230 235 240 Arg Asn Leu His Ile Pro Glu Val Cys Leu Lys Trp Glu Leu Met Asp 245 250 255 Glu Asp Tyr Lys Gly Arg Leu Cys Asn Pro Leu Asn Pro Phe Val Ser 260 265 270 His Lys Glu Ile Glu Ser Val Asn Ser Ala Val Val Met Pro Ala His 275 280 285 Gly Pro Ala Lys Ser Met Glu Tyr Asp Ala Val Ala Thr Thr His Ser 290 295 300 Trp Ile Pro Lys Arg Asn Arg Ser Ile Leu Asn Thr Ser Gln Arg Gly 305 310 315 320 Ile Leu Glu Asp Glu Gln Met Tyr Gln Lys Cys Cys Asn Leu Phe Glu 325 330 335 Lys Phe Phe Pro Ser Ser Ser Tyr Arg Lys Pro Val Gly Ile Ser Ser 340 345 350 Met Val Glu Ala Met Val Ser Arg Ala Arg Ile Asp Ala Arg Ile Asp 355 360 365 Phe Glu Ser Gly Arg Ile Lys Lys Asp Glu Phe Ala Glu Ile Met Lys 370 375 380 Ile Cys Ser Thr Ile Glu Glu Leu Arg Arg Gln Lys 385 390 395 93 1188 DNA Equine influenza virus H3N8 93 atgaaattga gaactcaaat accagcagaa atgctcgcaa gcattgatct gaaatatttc 60 aatgattcaa caaaaaagaa aattgagaag atacgaccac ttctggtcga tgggactgct 120 tcactgagtc ctggcatgat gatgggaatg ttcaacatgt tgagcactgt actaggtgta 180 tccatattaa acctgggcca gaggaaatac acaaagacca catactggtg ggatggtctg 240 caatcatccg atgattttgc tttgatagtg aatgcgccta atcatgaagg aatacaggct 300 ggagtagaca gattctatag aacttgcaaa ctggtcggga tcaacatgag caaaaagaag 360 tcctacataa atagaaccgg cacattcgaa ttcacaagct ttttctaccg gtatggtttt 420 gtcgccaatt tcagcatgga gctacccagt tttggggttt ccgggataaa tgaatctgca 480 gacatgagca ttggaatgac agttatcaaa aacaacatga taaataatga tctcggtccc 540 gccacggcac aaatggcact ccaactcttc attaaggatt atcggtacac ataccggtgt 600 caaagaggcg atacccagat acaaaccaga agatcctttg agttgaagaa actgtgggaa 660 cagactcgat caaagactgg tctactggta tcagatgggg gtccaaacct atacaacatc 720 agaaacctac acatcccgga agtctgtttg aaatgggagc tgatggatga agattataaa 780 gggaggctat gtaatccatt gaatcctttc gttagccaca aagaaattga atcagtgaac 840 agtgcagtag taatgcctgc gcatggccct gccaaaagca tggagtatga tgctgttgca 900 acaacacact cttggatccc caagaggaac cggtccatat tgaacacaag tcaaagggga 960 atactcgaag atgagcagat gtatcagaaa tgctgcaacc tgtttgaaaa attcttcccc 1020 agcagctcat acagaaaacc agtcggaatt tctagtatgg ttgaggccat ggtgtccagg 1080 gcccgcattg atgcacgaat tgacttcgaa tctggacgga taaagaagga tgagttcgct 1140 gagatcatga agatctgttc caccattgaa gagctcagac ggcaaaaa 1188 94 1241 DNA Equine influenza virus H3N8 CDS (8)..(1195) 94 caaaagt atg aaa ttg aga act caa ata cca gca gaa atg ctc gca agc 49 Met Lys Leu Arg Thr Gln Ile Pro Ala Glu Met Leu Ala Ser 1 5 10 att gat ctg aaa tat ttc aat gat tca aca aaa aag aaa att gag aag 97 Ile Asp Leu Lys Tyr Phe Asn Asp Ser Thr Lys Lys Lys Ile Glu Lys 15 20 25 30 ata cga cca ctt ctg gtc gat ggg act gct tca ctg agt cct ggc atg 145 Ile Arg Pro Leu Leu Val Asp Gly Thr Ala Ser Leu Ser Pro Gly Met 35 40 45 atg atg gga atg ttc aac atg ttg agc act gta cta ggt gta tcc ata 193 Met Met Gly Met Phe Asn Met Leu Ser Thr Val Leu Gly Val Ser Ile 50 55 60 tta aac ctg ggc cag agg aaa tac aca aag acc aca tac tgg tgg gat 241 Leu Asn Leu Gly Gln Arg Lys Tyr Thr Lys Thr Thr Tyr Trp Trp Asp 65 70 75 ggt ctg caa tca tcc gat gat ttt gct ttg ata gtg aat gcg cct aat 289 Gly Leu Gln Ser Ser Asp Asp Phe Ala Leu Ile Val Asn Ala Pro Asn 80 85 90 cat gaa gga ata cag gct gga gta gac aga ttc tat aga act tgc aaa 337 His Glu Gly Ile Gln Ala Gly Val Asp Arg Phe Tyr Arg Thr Cys Lys 95 100 105 110 ctg gtc ggg atc aac atg agc aaa aag aag tcc tac ata aat aga acc 385 Leu Val Gly Ile Asn Met Ser Lys Lys Lys Ser Tyr Ile Asn Arg Thr 115 120 125 ggc aca ttc gaa ttc aca agc ttt ttc tac cgg tat ggt ttt gtc gcc 433 Gly Thr Phe Glu Phe Thr Ser Phe Phe Tyr Arg Tyr Gly Phe Val Ala 130 135 140 aat ttc agc atg gag cta ccc agt ttt ggg gtt tcc ggg ata aat gaa 481 Asn Phe Ser Met Glu Leu Pro Ser Phe Gly Val Ser Gly Ile Asn Glu 145 150 155 tct gca gac atg agc att gga atg aca gtt atc aaa aac aac atg ata 529 Ser Ala Asp Met Ser Ile Gly Met Thr Val Ile Lys Asn Asn Met Ile 160 165 170 aat aat gat ctc ggt ccc gcc acg gca caa atg gca ctc caa ctc ttc 577 Asn Asn Asp Leu Gly Pro Ala Thr Ala Gln Met Ala Leu Gln Leu Phe 175 180 185 190 att aag gat tat cgg tac aca tac cgg tgt caa aga ggc gat acc cag 625 Ile Lys Asp Tyr Arg Tyr Thr Tyr Arg Cys Gln Arg Gly Asp Thr Gln 195 200 205 ata caa acc aga aga tcc ttt gag ttg aag aaa ctg tgg gaa cag act 673 Ile Gln Thr Arg Arg Ser Phe Glu Leu Lys Lys Leu Trp Glu Gln Thr 210 215 220 cga tca aag act ggt cta ctg gta tca gat ggg ggt cca aac cta tac 721 Arg Ser Lys Thr Gly Leu Leu Val Ser Asp Gly Gly Pro Asn Leu Tyr 225 230 235 aac atc aga aac cta cac atc ccg gaa gtc tgt ttg aaa tgg gag ctg 769 Asn Ile Arg Asn Leu His Ile Pro Glu Val Cys Leu Lys Trp Glu Leu 240 245 250 atg gat gaa gat tat aaa ggg agg cta tgt aat cca ttg aat cct ttc 817 Met Asp Glu Asp Tyr Lys Gly Arg Leu Cys Asn Pro Leu Asn Pro Phe 255 260 265 270 gtt agc cac aaa gaa att gaa tca gtg aac agt gca gta gta atg cct 865 Val Ser His Lys Glu Ile Glu Ser Val Asn Ser Ala Val Val Met Pro 275 280 285 gcg cat ggc cct gcc aaa agc atg gag tat gat gct gtt gca aca aca 913 Ala His Gly Pro Ala Lys Ser Met Glu Tyr Asp Ala Val Ala Thr Thr 290 295 300 cac tct tgg atc ccc aag agg aac cgg tcc ata ttg aac aca agt caa 961 His Ser Trp Ile Pro Lys Arg Asn Arg Ser Ile Leu Asn Thr Ser Gln 305 310 315 agg gga ata ctc gaa gat gag cag atg tat cag aaa tgc tgc aac ctg 1009 Arg Gly Ile Leu Glu Asp Glu Gln Met Tyr Gln Lys Cys Cys Asn Leu 320 325 330 ttt gaa aaa ttc ttc ccc agc agc tca tac aga aga cca gtc gga att 1057 Phe Glu Lys Phe Phe Pro Ser Ser Ser Tyr Arg Arg Pro Val Gly Ile 335 340 345 350 tct agt atg gtt gag gcc atg gtg tcc agg gcc cgc att gat gca cga 1105 Ser Ser Met Val Glu Ala Met Val Ser Arg Ala Arg Ile Asp Ala Arg 355 360 365 att gac ttc gaa tct gga cgg ata aag aag gat gag ttc gct gag atc 1153 Ile Asp Phe Glu Ser Gly Arg Ile Lys Lys Asp Glu Phe Ala Glu Ile 370 375 380 atg aag atc tgt tcc acc att gaa gag ctc aga cgg caa aaa 1195 Met Lys Ile Cys Ser Thr Ile Glu Glu Leu Arg Arg Gln Lys 385 390 395 tagtgaattt agcttgatct tcgtgaaaaa atgccttgtt tctact 1241 95 396 PRT Equine influenza virus H3N8 95 Met Lys Leu Arg Thr Gln Ile Pro Ala Glu Met Leu Ala Ser Ile Asp 1 5 10 15 Leu Lys Tyr Phe Asn Asp Ser Thr Lys Lys Lys Ile Glu Lys Ile Arg 20 25 30 Pro Leu Leu Val Asp Gly Thr Ala Ser Leu Ser Pro Gly Met Met Met 35 40 45 Gly Met Phe Asn Met Leu Ser Thr Val Leu Gly Val Ser Ile Leu Asn 50 55 60 Leu Gly Gln Arg Lys Tyr Thr Lys Thr Thr Tyr Trp Trp Asp Gly Leu 65 70 75 80 Gln Ser Ser Asp Asp Phe Ala Leu Ile Val Asn Ala Pro Asn His Glu 85 90 95 Gly Ile Gln Ala Gly Val Asp Arg Phe Tyr Arg Thr Cys Lys Leu Val 100 105 110 Gly Ile Asn Met Ser Lys Lys Lys Ser Tyr Ile Asn Arg Thr Gly Thr 115 120 125 Phe Glu Phe Thr Ser Phe Phe Tyr Arg Tyr Gly Phe Val Ala Asn Phe 130 135 140 Ser Met Glu Leu Pro Ser Phe Gly Val Ser Gly Ile Asn Glu Ser Ala 145 150 155 160 Asp Met Ser Ile Gly Met Thr Val Ile Lys Asn Asn Met Ile Asn Asn 165 170 175 Asp Leu Gly Pro Ala Thr Ala Gln Met Ala Leu Gln Leu Phe Ile Lys 180 185 190 Asp Tyr Arg Tyr Thr Tyr Arg Cys Gln Arg Gly Asp Thr Gln Ile Gln 195 200 205 Thr Arg Arg Ser Phe Glu Leu Lys Lys Leu Trp Glu Gln Thr Arg Ser 210 215 220 Lys Thr Gly Leu Leu Val Ser Asp Gly Gly Pro Asn Leu Tyr Asn Ile 225 230 235 240 Arg Asn Leu His Ile Pro Glu Val Cys Leu Lys Trp Glu Leu Met Asp 245 250 255 Glu Asp Tyr Lys Gly Arg Leu Cys Asn Pro Leu Asn Pro Phe Val Ser 260 265 270 His Lys Glu Ile Glu Ser Val Asn Ser Ala Val Val Met Pro Ala His 275 280 285 Gly Pro Ala Lys Ser Met Glu Tyr Asp Ala Val Ala Thr Thr His Ser 290 295 300 Trp Ile Pro Lys Arg Asn Arg Ser Ile Leu Asn Thr Ser Gln Arg Gly 305 310 315 320 Ile Leu Glu Asp Glu Gln Met Tyr Gln Lys Cys Cys Asn Leu Phe Glu 325 330 335 Lys Phe Phe Pro Ser Ser Ser Tyr Arg Arg Pro Val Gly Ile Ser Ser 340 345 350 Met Val Glu Ala Met Val Ser Arg Ala Arg Ile Asp Ala Arg Ile Asp 355 360 365 Phe Glu Ser Gly Arg Ile Lys Lys Asp Glu Phe Ala Glu Ile Met Lys 370 375 380 Ile Cys Ser Thr Ile Glu Glu Leu Arg Arg Gln Lys 385 390 395 96 1188 DNA Equine influenza virus H3N8 96 atgaaattga gaactcaaat accagcagaa atgctcgcaa gcattgatct gaaatatttc 60 aatgattcaa caaaaaagaa aattgagaag atacgaccac ttctggtcga tgggactgct 120 tcactgagtc ctggcatgat gatgggaatg ttcaacatgt tgagcactgt actaggtgta 180 tccatattaa acctgggcca gaggaaatac acaaagacca catactggtg ggatggtctg 240 caatcatccg atgattttgc tttgatagtg aatgcgccta atcatgaagg aatacaggct 300 ggagtagaca gattctatag aacttgcaaa ctggtcggga tcaacatgag caaaaagaag 360 tcctacataa atagaaccgg cacattcgaa ttcacaagct ttttctaccg gtatggtttt 420 gtcgccaatt tcagcatgga gctacccagt tttggggttt ccgggataaa tgaatctgca 480 gacatgagca ttggaatgac agttatcaaa aacaacatga taaataatga tctcggtccc 540 gccacggcac aaatggcact ccaactcttc attaaggatt atcggtacac ataccggtgt 600 caaagaggcg atacccagat acaaaccaga agatcctttg agttgaagaa actgtgggaa 660 cagactcgat caaagactgg tctactggta tcagatgggg gtccaaacct atacaacatc 720 agaaacctac acatcccgga agtctgtttg aaatgggagc tgatggatga agattataaa 780 gggaggctat gtaatccatt gaatcctttc gttagccaca aagaaattga atcagtgaac 840 agtgcagtag taatgcctgc gcatggccct gccaaaagca tggagtatga tgctgttgca 900 acaacacact cttggatccc caagaggaac cggtccatat tgaacacaag tcaaagggga 960 atactcgaag atgagcagat gtatcagaaa tgctgcaacc tgtttgaaaa attcttcccc 1020 agcagctcat acagaagacc agtcggaatt tctagtatgg ttgaggccat ggtgtccagg 1080 gcccgcattg atgcacgaat tgacttcgaa tctggacgga taaagaagga tgagttcgct 1140 gagatcatga agatctgttc caccattgaa gagctcagac ggcaaaaa 1188 97 20 DNA Artificial sequence Synthetic Primer 97 taaatagaac cggcacattc 20 98 17 DNA Artificial sequence Synthetic Primer 98 caaagaaatt gaatcag 17 99 18 DNA Artificial sequence Synthetic Primer 99 caagcattac tactgcac 18 100 19 DNA Artificial sequence Synthetic Primer 100 agtctgttcc cacagtttc 19 101 20 DNA Artificial sequence Synthetic Primer 101 gaattcgaat gtgccggttc 20 102 20 DNA Artificial sequence Synthetic Primer 102 aaaacaagga ttttttcacg 20 103 2341 DNA Equine influenza virus H3N8 CDS (25)..(2295) 103 agcaaaagca ggcaaactat ttga atg gat gtc aat ccg act cta ctc ttc 51 Met Asp Val Asn Pro Thr Leu Leu Phe 1 5 tta aag gtg cca gcg caa aat gct ata agc aca aca ttc cct tat act 99 Leu Lys Val Pro Ala Gln Asn Ala Ile Ser Thr Thr Phe Pro Tyr Thr 10 15 20 25 gga gat cct ccc tac agt cat gga aca ggg aca gga tac acc atg gat 147 Gly Asp Pro Pro Tyr Ser His Gly Thr Gly Thr Gly Tyr Thr Met Asp 30 35 40 act gtc aac aga aca cat caa tac tca gaa aag ggg aaa tgg aca aca 195 Thr Val Asn Arg Thr His Gln Tyr Ser Glu Lys Gly Lys Trp Thr Thr 45 50 55 aac act gag att gga gca cca caa ctt aat cca atc gat gga ccg ctt 243 Asn Thr Glu Ile Gly Ala Pro Gln Leu Asn Pro Ile Asp Gly Pro Leu 60 65 70 cct gaa gac aat gaa cca agt ggg tac gcc caa aca gat tgt gta ttg 291 Pro Glu Asp Asn Glu Pro Ser Gly Tyr Ala Gln Thr Asp Cys Val Leu 75 80 85 gaa gca atg gct ttc ctt gaa gaa tcc cat ccc gga atc ttt gaa aat 339 Glu Ala Met Ala Phe Leu Glu Glu Ser His Pro Gly Ile Phe Glu Asn 90 95 100 105 tcg tgt ctt gaa aca atg gag gtg gtt cag cag aca aga gtg gac aaa 387 Ser Cys Leu Glu Thr Met Glu Val Val Gln Gln Thr Arg Val Asp Lys 110 115 120 cta aca caa ggc cga caa act tac gat tgg acc ttg aat agg aat caa 435 Leu Thr Gln Gly Arg Gln Thr Tyr Asp Trp Thr Leu Asn Arg Asn Gln 125 130 135 cct gcc gca aca gca ctt gct aat aca att gaa gtg ttc aga tca aat 483 Pro Ala Ala Thr Ala Leu Ala Asn Thr Ile Glu Val Phe Arg Ser Asn 140 145 150 gat ctg act tcc agt gag tca ggg aga tta atg gac ttc ctc aaa gat 531 Asp Leu Thr Ser Ser Glu Ser Gly Arg Leu Met Asp Phe Leu Lys Asp 155 160 165 gtc atg gag tcc atg aac aag gaa gaa atg gaa ata aca aca cac ttc 579 Val Met Glu Ser Met Asn Lys Glu Glu Met Glu Ile Thr Thr His Phe 170 175 180 185 caa cgg aag aga aga gta aga gac aac atg aca aag aga atg gtg aca 627 Gln Arg Lys Arg Arg Val Arg Asp Asn Met Thr Lys Arg Met Val Thr 190 195 200 cag aga acc ata ggg aag aaa aaa caa cga tta aac aga aag agc tat 675 Gln Arg Thr Ile Gly Lys Lys Lys Gln Arg Leu Asn Arg Lys Ser Tyr 205 210 215 ctg atc agg gca tta acc tta aac aca atg acc aag gac gct gag aga 723 Leu Ile Arg Ala Leu Thr Leu Asn Thr Met Thr Lys Asp Ala Glu Arg 220 225 230 ggg aaa ttg aaa cga cga gca att gca acc cca gga atg cag ata aga 771 Gly Lys Leu Lys Arg Arg Ala Ile Ala Thr Pro Gly Met Gln Ile Arg 235 240 245 ggg ttt gta tat ttt gtt gaa aca tta gcc cga aga ata tgt gaa aag 819 Gly Phe Val Tyr Phe Val Glu Thr Leu Ala Arg Arg Ile Cys Glu Lys 250 255 260 265 ctt gaa caa tca gga ttg cca gtt ggc ggt aat gag aaa aag gcc aaa 867 Leu Glu Gln Ser Gly Leu Pro Val Gly Gly Asn Glu Lys Lys Ala Lys 270 275 280 ctg gct aat gtc gtc aga aaa atg atg act aat tcc caa gac act gaa 915 Leu Ala Asn Val Val Arg Lys Met Met Thr Asn Ser Gln Asp Thr Glu 285 290 295 ctc tcc ttc acc atc act ggg gac aat acc aaa tgg aat gaa aat cag 963 Leu Ser Phe Thr Ile Thr Gly Asp Asn Thr Lys Trp Asn Glu Asn Gln 300 305 310 aac cca cgc atg ttc ctg gca atg atc aca tac ata act aga aac cag 1011 Asn Pro Arg Met Phe Leu Ala Met Ile Thr Tyr Ile Thr Arg Asn Gln 315 320 325 cca gaa tgg ttc aga aat gtt cta agc att gca ccg att atg ttc tca 1059 Pro Glu Trp Phe Arg Asn Val Leu Ser Ile Ala Pro Ile Met Phe Ser 330 335 340 345 aat aaa atg gca aga ctg ggg aaa gga tat atg ttt gaa agc aaa agt 1107 Asn Lys Met Ala Arg Leu Gly Lys Gly Tyr Met Phe Glu Ser Lys Ser 350 355 360 atg aaa ttg aga act caa ata cca gca gaa atg ctc gca agc att gat 1155 Met Lys Leu Arg Thr Gln Ile Pro Ala Glu Met Leu Ala Ser Ile Asp 365 370 375 ctg aaa tat ttc aat gat tca aca aaa aag aaa att gag aag ata cga 1203 Leu Lys Tyr Phe Asn Asp Ser Thr Lys Lys Lys Ile Glu Lys Ile Arg 380 385 390 cca ctt ctg gtc gat ggg act gct tca ctg agt cct ggc atg atg atg 1251 Pro Leu Leu Val Asp Gly Thr Ala Ser Leu Ser Pro Gly Met Met Met 395 400 405 gga atg ttc aac atg ttg agc act gta cta ggt gta tcc ata tta aac 1299 Gly Met Phe Asn Met Leu Ser Thr Val Leu Gly Val Ser Ile Leu Asn 410 415 420 425 ctg ggc cag agg aaa tac aca aag acc aca tac tgg tgg gat ggt ctg 1347 Leu Gly Gln Arg Lys Tyr Thr Lys Thr Thr Tyr Trp Trp Asp Gly Leu 430 435 440 caa tca tcc gat gat ttt gct ttg ata gtg aat gcg cct aat cat gaa 1395 Gln Ser Ser Asp Asp Phe Ala Leu Ile Val Asn Ala Pro Asn His Glu 445 450 455 gga ata cag gct gga gta gac aga ttc tat aga act tgc aaa ctg gtc 1443 Gly Ile Gln Ala Gly Val Asp Arg Phe Tyr Arg Thr Cys Lys Leu Val 460 465 470 ggg atc aac atg agc aaa aag aag tcc tac ata aat aga acc ggc wca 1491 Gly Ile Asn Met Ser Lys Lys Lys Ser Tyr Ile Asn Arg Thr Gly Xaa 475 480 485 ttc gaa ttc aca agc ttt ttc tac cgg tat ggt ttt gtc gcc aat ttc 1539 Phe Glu Phe Thr Ser Phe Phe Tyr Arg Tyr Gly Phe Val Ala Asn Phe 490 495 500 505 agc atg gag cta ccc agt ttt ggg gtt tcc ggg ata aat gaa tct gca 1587 Ser Met Glu Leu Pro Ser Phe Gly Val Ser Gly Ile Asn Glu Ser Ala 510 515 520 gac atg agc att gga atg aca gtt atc aaa aac aac atg ata aat aat 1635 Asp Met Ser Ile Gly Met Thr Val Ile Lys Asn Asn Met Ile Asn Asn 525 530 535 gat ctc ggt ccc gcc acg gca caa atg gca ctc caa ctc ttc att aag 1683 Asp Leu Gly Pro Ala Thr Ala Gln Met Ala Leu Gln Leu Phe Ile Lys 540 545 550 gat tat cgg tac aca tac cgg tgc cat aga ggc gat acc cag ata caa 1731 Asp Tyr Arg Tyr Thr Tyr Arg Cys His Arg Gly Asp Thr Gln Ile Gln 555 560 565 acc aga aga tcc ttt gag ttg aag aaa ctg tgg gaa cag act cga tca 1779 Thr Arg Arg Ser Phe Glu Leu Lys Lys Leu Trp Glu Gln Thr Arg Ser 570 575 580 585 aag act ggt cta ctg gta tca gat ggg ggt cca aac cta tac aac atc 1827 Lys Thr Gly Leu Leu Val Ser Asp Gly Gly Pro Asn Leu Tyr Asn Ile 590 595 600 aga aac cta cac atc ccg gaa gtc tgt ttg aaa tgg gag ctg atg gat 1875 Arg Asn Leu His Ile Pro Glu Val Cys Leu Lys Trp Glu Leu Met Asp 605 610 615 gaa gat tat aaa ggg agg cta tgt aat cca ttg aat cct ttc gtt agc 1923 Glu Asp Tyr Lys Gly Arg Leu Cys Asn Pro Leu Asn Pro Phe Val Ser 620 625 630 cac aaa gaa att gaa tca gtg aac agt gca gta gta atg cct gcg cat 1971 His Lys Glu Ile Glu Ser Val Asn Ser Ala Val Val Met Pro Ala His 635 640 645 ggc cct gcc aaa agc atg gag tat gat gct gtt gca aca aca cac tct 2019 Gly Pro Ala Lys Ser Met Glu Tyr Asp Ala Val Ala Thr Thr His Ser 650 655 660 665 tgg atc ccc aag agg aac cgg tcc ata ttg aac aca agt caa agg gga 2067 Trp Ile Pro Lys Arg Asn Arg Ser Ile Leu Asn Thr Ser Gln Arg Gly 670 675 680 ata ctc gaa gat gag cag atg tat cag aaa tgc tgc aac ctg ttt gaa 2115 Ile Leu Glu Asp Glu Gln Met Tyr Gln Lys Cys Cys Asn Leu Phe Glu 685 690 695 aaa ttc ttc ccc agc agc tca tac aga aga cca gtc gga att tct agt 2163 Lys Phe Phe Pro Ser Ser Ser Tyr Arg Arg Pro Val Gly Ile Ser Ser 700 705 710 atg gtt gag gcc atg gtg tcc agg gcc cgc att gat gca cga att gac 2211 Met Val Glu Ala Met Val Ser Arg Ala Arg Ile Asp Ala Arg Ile Asp 715 720 725 ttc gaa tct gga cgg ata aag aag gat gag ttc gct gag atc atg aag 2259 Phe Glu Ser Gly Arg Ile Lys Lys Asp Glu Phe Ala Glu Ile Met Lys 730 735 740 745 atc tgt tcc acc att gaa gag ctc aga cgg caa aaa tagtgaattt 2305 Ile Cys Ser Thr Ile Glu Glu Leu Arg Arg Gln Lys 750 755 agcttgatct tcgtgaaaaa atgccttgtt tctact 2341 104 757 PRT Equine influenza virus H3N8 misc_feature (489)..(489) The ′Xaa′ at location 489 stands for Thr or Ser 104 Met Asp Val Asn Pro Thr Leu Leu Phe Leu Lys Val Pro Ala Gln Asn 1 5 10 15 Ala Ile Ser Thr Thr Phe Pro Tyr Thr Gly Asp Pro Pro Tyr Ser His 20 25 30 Gly Thr Gly Thr Gly Tyr Thr Met Asp Thr Val Asn Arg Thr His Gln 35 40 45 Tyr Ser Glu Lys Gly Lys Trp Thr Thr Asn Thr Glu Ile Gly Ala Pro 50 55 60 Gln Leu Asn Pro Ile Asp Gly Pro Leu Pro Glu Asp Asn Glu Pro Ser 65 70 75 80 Gly Tyr Ala Gln Thr Asp Cys Val Leu Glu Ala Met Ala Phe Leu Glu 85 90 95 Glu Ser His Pro Gly Ile Phe Glu Asn Ser Cys Leu Glu Thr Met Glu 100 105 110 Val Val Gln Gln Thr Arg Val Asp Lys Leu Thr Gln Gly Arg Gln Thr 115 120 125 Tyr Asp Trp Thr Leu Asn Arg Asn Gln Pro Ala Ala Thr Ala Leu Ala 130 135 140 Asn Thr Ile Glu Val Phe Arg Ser Asn Asp Leu Thr Ser Ser Glu Ser 145 150 155 160 Gly Arg Leu Met Asp Phe Leu Lys Asp Val Met Glu Ser Met Asn Lys 165 170 175 Glu Glu Met Glu Ile Thr Thr His Phe Gln Arg Lys Arg Arg Val Arg 180 185 190 Asp Asn Met Thr Lys Arg Met Val Thr Gln Arg Thr Ile Gly Lys Lys 195 200 205 Lys Gln Arg Leu Asn Arg Lys Ser Tyr Leu Ile Arg Ala Leu Thr Leu 210 215 220 Asn Thr Met Thr Lys Asp Ala Glu Arg Gly Lys Leu Lys Arg Arg Ala 225 230 235 240 Ile Ala Thr Pro Gly Met Gln Ile Arg Gly Phe Val Tyr Phe Val Glu 245 250 255 Thr Leu Ala Arg Arg Ile Cys Glu Lys Leu Glu Gln Ser Gly Leu Pro 260 265 270 Val Gly Gly Asn Glu Lys Lys Ala Lys Leu Ala Asn Val Val Arg Lys 275 280 285 Met Met Thr Asn Ser Gln Asp Thr Glu Leu Ser Phe Thr Ile Thr Gly 290 295 300 Asp Asn Thr Lys Trp Asn Glu Asn Gln Asn Pro Arg Met Phe Leu Ala 305 310 315 320 Met Ile Thr Tyr Ile Thr Arg Asn Gln Pro Glu Trp Phe Arg Asn Val 325 330 335 Leu Ser Ile Ala Pro Ile Met Phe Ser Asn Lys Met Ala Arg Leu Gly 340 345 350 Lys Gly Tyr Met Phe Glu Ser Lys Ser Met Lys Leu Arg Thr Gln Ile 355 360 365 Pro Ala Glu Met Leu Ala Ser Ile Asp Leu Lys Tyr Phe Asn Asp Ser 370 375 380 Thr Lys Lys Lys Ile Glu Lys Ile Arg Pro Leu Leu Val Asp Gly Thr 385 390 395 400 Ala Ser Leu Ser Pro Gly Met Met Met Gly Met Phe Asn Met Leu Ser 405 410 415 Thr Val Leu Gly Val Ser Ile Leu Asn Leu Gly Gln Arg Lys Tyr Thr 420 425 430 Lys Thr Thr Tyr Trp Trp Asp Gly Leu Gln Ser Ser Asp Asp Phe Ala 435 440 445 Leu Ile Val Asn Ala Pro Asn His Glu Gly Ile Gln Ala Gly Val Asp 450 455 460 Arg Phe Tyr Arg Thr Cys Lys Leu Val Gly Ile Asn Met Ser Lys Lys 465 470 475 480 Lys Ser Tyr Ile Asn Arg Thr Gly Xaa Phe Glu Phe Thr Ser Phe Phe 485 490 495 Tyr Arg Tyr Gly Phe Val Ala Asn Phe Ser Met Glu Leu Pro Ser Phe 500 505 510 Gly Val Ser Gly Ile Asn Glu Ser Ala Asp Met Ser Ile Gly Met Thr 515 520 525 Val Ile Lys Asn Asn Met Ile Asn Asn Asp Leu Gly Pro Ala Thr Ala 530 535 540 Gln Met Ala Leu Gln Leu Phe Ile Lys Asp Tyr Arg Tyr Thr Tyr Arg 545 550 555 560 Cys His Arg Gly Asp Thr Gln Ile Gln Thr Arg Arg Ser Phe Glu Leu 565 570 575 Lys Lys Leu Trp Glu Gln Thr Arg Ser Lys Thr Gly Leu Leu Val Ser 580 585 590 Asp Gly Gly Pro Asn Leu Tyr Asn Ile Arg Asn Leu His Ile Pro Glu 595 600 605 Val Cys Leu Lys Trp Glu Leu Met Asp Glu Asp Tyr Lys Gly Arg Leu 610 615 620 Cys Asn Pro Leu Asn Pro Phe Val Ser His Lys Glu Ile Glu Ser Val 625 630 635 640 Asn Ser Ala Val Val Met Pro Ala His Gly Pro Ala Lys Ser Met Glu 645 650 655 Tyr Asp Ala Val Ala Thr Thr His Ser Trp Ile Pro Lys Arg Asn Arg 660 665 670 Ser Ile Leu Asn Thr Ser Gln Arg Gly Ile Leu Glu Asp Glu Gln Met 675 680 685 Tyr Gln Lys Cys Cys Asn Leu Phe Glu Lys Phe Phe Pro Ser Ser Ser 690 695 700 Tyr Arg Arg Pro Val Gly Ile Ser Ser Met Val Glu Ala Met Val Ser 705 710 715 720 Arg Ala Arg Ile Asp Ala Arg Ile Asp Phe Glu Ser Gly Arg Ile Lys 725 730 735 Lys Asp Glu Phe Ala Glu Ile Met Lys Ile Cys Ser Thr Ile Glu Glu 740 745 750 Leu Arg Arg Gln Lys 755 105 2271 DNA Equine influenza virus H3N8 105 atggatgtca atccgactct actcttctta aaggtgccag cgcaaaatgc tataagcaca 60 acattccctt atactggaga tcctccctac agtcatggaa cagggacagg atacaccatg 120 gatactgtca acagaacaca tcaatactca gaaaagggga aatggacaac aaacactgag 180 attggagcac cacaacttaa tccaatcgat ggaccgcttc ctgaagacaa tgaaccaagt 240 gggtacgccc aaacagattg tgtattggaa gcaatggctt tccttgaaga atcccatccc 300 ggaatctttg aaaattcgtg tcttgaaaca atggaggtgg ttcagcagac aagagtggac 360 aaactaacac aaggccgaca aacttacgat tggaccttga ataggaatca acctgccgca 420 acagcacttg ctaatacaat tgaagtgttc agatcaaatg atctgacttc cagtgagtca 480 gggagattaa tggacttcct caaagatgtc atggagtcca tgaacaagga agaaatggaa 540 ataacaacac acttccaacg gaagagaaga gtaagagaca acatgacaaa gagaatggtg 600 acacagagaa ccatagggaa gaaaaaacaa cgattaaaca gaaagagcta tctgatcagg 660 gcattaacct taaacacaat gaccaaggac gctgagagag ggaaattgaa acgacgagca 720 attgcaaccc caggaatgca gataagaggg tttgtatatt ttgttgaaac attagcccga 780 agaatatgtg aaaagcttga acaatcagga ttgccagttg gcggtaatga gaaaaaggcc 840 aaactggcta atgtcgtcag aaaaatgatg actaattccc aagacactga actctccttc 900 accatcactg gggacaatac caaatggaat gaaaatcaga acccacgcat gttcctggca 960 atgatcacat acataactag aaaccagcca gaatggttca gaaatgttct aagcattgca 1020 ccgattatgt tctcaaataa aatggcaaga ctggggaaag gatatatgtt tgaaagcaaa 1080 agtatgaaat tgagaactca aataccagca gaaatgctcg caagcattga tctgaaatat 1140 ttcaatgatt caacaaaaaa gaaaattgag aagatacgac cacttctggt cgatgggact 1200 gcttcactga gtcctggcat gatgatggga atgttcaaca tgttgagcac tgtactaggt 1260 gtatccatat taaacctggg ccagaggaaa tacacaaaga ccacatactg gtgggatggt 1320 ctgcaatcat ccgatgattt tgctttgata gtgaatgcgc ctaatcatga aggaatacag 1380 gctggagtag acagattcta tagaacttgc aaactggtcg ggatcaacat gagcaaaaag 1440 aagtcctaca taaatagaac cggcwcattc gaattcacaa gctttttcta ccggtatggt 1500 tttgtcgcca atttcagcat ggagctaccc agttttgggg tttccgggat aaatgaatct 1560 gcagacatga gcattggaat gacagttatc aaaaacaaca tgataaataa tgatctcggt 1620 cccgccacgg cacaaatggc actccaactc ttcattaagg attatcggta cacataccgg 1680 tgccatagag gcgataccca gatacaaacc agaagatcct ttgagttgaa gaaactgtgg 1740 gaacagactc gatcaaagac tggtctactg gtatcagatg ggggtccaaa cctatacaac 1800 atcagaaacc tacacatccc ggaagtctgt ttgaaatggg agctgatgga tgaagattat 1860 aaagggaggc tatgtaatcc attgaatcct ttcgttagcc acaaagaaat tgaatcagtg 1920 aacagtgcag tagtaatgcc tgcgcatggc cctgccaaaa gcatggagta tgatgctgtt 1980 gcaacaacac actcttggat ccccaagagg aaccggtcca tattgaacac aagtcaaagg 2040 ggaatactcg aagatgagca gatgtatcag aaatgctgca acctgtttga aaaattcttc 2100 cccagcagct catacagaag accagtcgga atttctagta tggttgaggc catggtgtcc 2160 agggcccgca ttgatgcacg aattgacttc gaatctggac ggataaagaa ggatgagttc 2220 gctgagatca tgaagatctg ttccaccatt gaagagctca gacggcaaaa a 2271 106 2341 DNA Equine influenza virus H3N8 CDS (25)..(2295) 106 agcaaaagca ggcaaactat ttga atg gat gtc aat ccg act cta ctc ttc 51 Met Asp Val Asn Pro Thr Leu Leu Phe 1 5 tta aag gtg cca gcg caa aat gct ata agc aca aca ttc cct tat act 99 Leu Lys Val Pro Ala Gln Asn Ala Ile Ser Thr Thr Phe Pro Tyr Thr 10 15 20 25 gga gat cct ccc tac agt cat gga aca ggg aca gga tac acc atg gat 147 Gly Asp Pro Pro Tyr Ser His Gly Thr Gly Thr Gly Tyr Thr Met Asp 30 35 40 act gtc aac aga aca cat caa tac tca gaa aag ggg aaa tgg aca aca 195 Thr Val Asn Arg Thr His Gln Tyr Ser Glu Lys Gly Lys Trp Thr Thr 45 50 55 aac act gag att gga gca cca caa ctt aat cca atc gat gga ccg ctt 243 Asn Thr Glu Ile Gly Ala Pro Gln Leu Asn Pro Ile Asp Gly Pro Leu 60 65 70 cct gaa gac aat gaa cca agt ggg tac gcc caa aca gat tgt gta ttg 291 Pro Glu Asp Asn Glu Pro Ser Gly Tyr Ala Gln Thr Asp Cys Val Leu 75 80 85 gaa gca atg gct ttc ctt gaa gaa tcc cat ccc gga atc ttt gaa aat 339 Glu Ala Met Ala Phe Leu Glu Glu Ser His Pro Gly Ile Phe Glu Asn 90 95 100 105 tcg tgt ctt gaa aca atg gag gtg gtt cag cag aca aga gtg gac aaa 387 Ser Cys Leu Glu Thr Met Glu Val Val Gln Gln Thr Arg Val Asp Lys 110 115 120 cta aca caa ggc cga caa act tac gat tgg acc ttg aat agg aat caa 435 Leu Thr Gln Gly Arg Gln Thr Tyr Asp Trp Thr Leu Asn Arg Asn Gln 125 130 135 cct gcc gca aca gca ctt gct aat aca att gaa gtg ttc aga tca aat 483 Pro Ala Ala Thr Ala Leu Ala Asn Thr Ile Glu Val Phe Arg Ser Asn 140 145 150 gat ctg act tcc agt gag tca ggg aga tta atg gac ttc ctc aaa gat 531 Asp Leu Thr Ser Ser Glu Ser Gly Arg Leu Met Asp Phe Leu Lys Asp 155 160 165 gtc atg gag tcc atg aac aag gaa gaa atg gaa ata aca aca cac ttc 579 Val Met Glu Ser Met Asn Lys Glu Glu Met Glu Ile Thr Thr His Phe 170 175 180 185 caa cgg aag aga aga gta aga gac aac atg aca aag aga atg gtg aca 627 Gln Arg Lys Arg Arg Val Arg Asp Asn Met Thr Lys Arg Met Val Thr 190 195 200 cag aga acc ata ggg aag aaa aaa caa cga tta aac aga aag agc tat 675 Gln Arg Thr Ile Gly Lys Lys Lys Gln Arg Leu Asn Arg Lys Ser Tyr 205 210 215 ctg atc agg gca tta acc tta aac aca atg acc aag gac gct gag aga 723 Leu Ile Arg Ala Leu Thr Leu Asn Thr Met Thr Lys Asp Ala Glu Arg 220 225 230 ggg aaa ttg aaa cga cga gca att gca acc cca gga atg cag ata aga 771 Gly Lys Leu Lys Arg Arg Ala Ile Ala Thr Pro Gly Met Gln Ile Arg 235 240 245 ggg ttt gta tat ttt gtt gaa aca tta gcc cga aga ata tgt gaa aag 819 Gly Phe Val Tyr Phe Val Glu Thr Leu Ala Arg Arg Ile Cys Glu Lys 250 255 260 265 ctt gaa caa tca gga ttg cca gtt ggc ggt aat gag aaa aag gcc aaa 867 Leu Glu Gln Ser Gly Leu Pro Val Gly Gly Asn Glu Lys Lys Ala Lys 270 275 280 ctg gct aat gtc gtc aga aaa atg atg act aat tcc caa gac act gaa 915 Leu Ala Asn Val Val Arg Lys Met Met Thr Asn Ser Gln Asp Thr Glu 285 290 295 ctc tcc ttc acc atc act ggg gac aat acc aaa tgg aat gaa aat cag 963 Leu Ser Phe Thr Ile Thr Gly Asp Asn Thr Lys Trp Asn Glu Asn Gln 300 305 310 aac cca cgc atg ttc ctg gca atg atc aca tac ata act aga aac cag 1011 Asn Pro Arg Met Phe Leu Ala Met Ile Thr Tyr Ile Thr Arg Asn Gln 315 320 325 cca gaa tgg ttc aga aat gtt cta agc att gca ccg att atg ttc tca 1059 Pro Glu Trp Phe Arg Asn Val Leu Ser Ile Ala Pro Ile Met Phe Ser 330 335 340 345 aat aaa atg gca aga ctg ggg aaa gga tat atg ttt gaa agc aaa agt 1107 Asn Lys Met Ala Arg Leu Gly Lys Gly Tyr Met Phe Glu Ser Lys Ser 350 355 360 atg aaa ttg aga act caa ata cca gca gaa atg ctc gca agc att gat 1155 Met Lys Leu Arg Thr Gln Ile Pro Ala Glu Met Leu Ala Ser Ile Asp 365 370 375 ctg aaa tat ttc aat gat tca aca aaa aag aaa att gag aag ata cga 1203 Leu Lys Tyr Phe Asn Asp Ser Thr Lys Lys Lys Ile Glu Lys Ile Arg 380 385 390 cca ctt ctg gtc gat ggg act gct tca ctg agt cct ggc atg atg atg 1251 Pro Leu Leu Val Asp Gly Thr Ala Ser Leu Ser Pro Gly Met Met Met 395 400 405 gga atg ttc aac atg ttg agc act gta cta ggt gta tcc ata tta aac 1299 Gly Met Phe Asn Met Leu Ser Thr Val Leu Gly Val Ser Ile Leu Asn 410 415 420 425 ctg ggc cag agg aaa tac aca aag acc aca tac tgg tgg gat ggt ctg 1347 Leu Gly Gln Arg Lys Tyr Thr Lys Thr Thr Tyr Trp Trp Asp Gly Leu 430 435 440 caa tca tcc gat gat ttt gct ttg ata gtg aat gcg cct aat cat gaa 1395 Gln Ser Ser Asp Asp Phe Ala Leu Ile Val Asn Ala Pro Asn His Glu 445 450 455 gga ata cag gct gga gta gac aga ttc tat aga act tgc aaa ctg gtc 1443 Gly Ile Gln Ala Gly Val Asp Arg Phe Tyr Arg Thr Cys Lys Leu Val 460 465 470 ggg atc aac atg agc aaa aag aag tcc tac ata aat aga acc ggc aca 1491 Gly Ile Asn Met Ser Lys Lys Lys Ser Tyr Ile Asn Arg Thr Gly Thr 475 480 485 ttc gaa ttc aca agc ttt ttc tac cgg tat ggt ttt gtc gcc aat ttc 1539 Phe Glu Phe Thr Ser Phe Phe Tyr Arg Tyr Gly Phe Val Ala Asn Phe 490 495 500 505 agc atg gag cta ccc agt ttt ggg gtt tcc ggg ata aat gaa tct gca 1587 Ser Met Glu Leu Pro Ser Phe Gly Val Ser Gly Ile Asn Glu Ser Ala 510 515 520 gac atg agc att gga atg aca gtt atc aaa aac aac atg ata aat aat 1635 Asp Met Ser Ile Gly Met Thr Val Ile Lys Asn Asn Met Ile Asn Asn 525 530 535 gat ctc ggt ccc gcc acg gca caa atg gca ctc caa ctc ttc att aag 1683 Asp Leu Gly Pro Ala Thr Ala Gln Met Ala Leu Gln Leu Phe Ile Lys 540 545 550 gat tat cgg tac aca tac cgg tgt caa aga ggc gat acc cag ata caa 1731 Asp Tyr Arg Tyr Thr Tyr Arg Cys Gln Arg Gly Asp Thr Gln Ile Gln 555 560 565 acc aga aga tcc ttt gag ttg aag aaa ctg tgg gaa cag act cga tca 1779 Thr Arg Arg Ser Phe Glu Leu Lys Lys Leu Trp Glu Gln Thr Arg Ser 570 575 580 585 aag act ggt cta ctg gta tca gat ggg ggt cca aac cta tac aac atc 1827 Lys Thr Gly Leu Leu Val Ser Asp Gly Gly Pro Asn Leu Tyr Asn Ile 590 595 600 aga aac cta cac atc ccg gaa gtc tgt ttg aaa tgg gag ctg atg gat 1875 Arg Asn Leu His Ile Pro Glu Val Cys Leu Lys Trp Glu Leu Met Asp 605 610 615 gaa gat tat aaa ggg agg cta tgt aat cca ttg aat cct ttc gtt agc 1923 Glu Asp Tyr Lys Gly Arg Leu Cys Asn Pro Leu Asn Pro Phe Val Ser 620 625 630 cac aaa gaa att gaa tca gtg aac agt gca gta gta atg cct gcg cat 1971 His Lys Glu Ile Glu Ser Val Asn Ser Ala Val Val Met Pro Ala His 635 640 645 ggc cct gcc aaa agc atg gag tat gat gct gtt gca aca aca cac tct 2019 Gly Pro Ala Lys Ser Met Glu Tyr Asp Ala Val Ala Thr Thr His Ser 650 655 660 665 tgg atc ccc aag agg aac cgg tcc ata ttg aac aca agt caa agg gga 2067 Trp Ile Pro Lys Arg Asn Arg Ser Ile Leu Asn Thr Ser Gln Arg Gly 670 675 680 ata ctc gaa gat gag cag atg tat cag aaa tgc tgc aac ctg ttt gaa 2115 Ile Leu Glu Asp Glu Gln Met Tyr Gln Lys Cys Cys Asn Leu Phe Glu 685 690 695 aaa ttc ttc ccc agc agc tca tac aga ara cca gtc gga att tct agt 2163 Lys Phe Phe Pro Ser Ser Ser Tyr Arg Xaa Pro Val Gly Ile Ser Ser 700 705 710 atg gtt gag gcc atg gtg tcc agg gcc cgc att gat gca cga att gac 2211 Met Val Glu Ala Met Val Ser Arg Ala Arg Ile Asp Ala Arg Ile Asp 715 720 725 ttc gaa tct gga cgg ata aag aag gat gag ttc gct gag atc atg aag 2259 Phe Glu Ser Gly Arg Ile Lys Lys Asp Glu Phe Ala Glu Ile Met Lys 730 735 740 745 atc tgt tcc acc att gaa gag ctc aga cgg caa aaa tagtgaattt 2305 Ile Cys Ser Thr Ile Glu Glu Leu Arg Arg Gln Lys 750 755 agcttgatct tcgtgaaaaa atgccttgtt tctact 2341 107 757 PRT Equine influenza virus H3N8 misc_feature (707)..(707) The ′Xaa′ at location 707 stands for Arg, or Lys. 107 Met Asp Val Asn Pro Thr Leu Leu Phe Leu Lys Val Pro Ala Gln Asn 1 5 10 15 Ala Ile Ser Thr Thr Phe Pro Tyr Thr Gly Asp Pro Pro Tyr Ser His 20 25 30 Gly Thr Gly Thr Gly Tyr Thr Met Asp Thr Val Asn Arg Thr His Gln 35 40 45 Tyr Ser Glu Lys Gly Lys Trp Thr Thr Asn Thr Glu Ile Gly Ala Pro 50 55 60 Gln Leu Asn Pro Ile Asp Gly Pro Leu Pro Glu Asp Asn Glu Pro Ser 65 70 75 80 Gly Tyr Ala Gln Thr Asp Cys Val Leu Glu Ala Met Ala Phe Leu Glu 85 90 95 Glu Ser His Pro Gly Ile Phe Glu Asn Ser Cys Leu Glu Thr Met Glu 100 105 110 Val Val Gln Gln Thr Arg Val Asp Lys Leu Thr Gln Gly Arg Gln Thr 115 120 125 Tyr Asp Trp Thr Leu Asn Arg Asn Gln Pro Ala Ala Thr Ala Leu Ala 130 135 140 Asn Thr Ile Glu Val Phe Arg Ser Asn Asp Leu Thr Ser Ser Glu Ser 145 150 155 160 Gly Arg Leu Met Asp Phe Leu Lys Asp Val Met Glu Ser Met Asn Lys 165 170 175 Glu Glu Met Glu Ile Thr Thr His Phe Gln Arg Lys Arg Arg Val Arg 180 185 190 Asp Asn Met Thr Lys Arg Met Val Thr Gln Arg Thr Ile Gly Lys Lys 195 200 205 Lys Gln Arg Leu Asn Arg Lys Ser Tyr Leu Ile Arg Ala Leu Thr Leu 210 215 220 Asn Thr Met Thr Lys Asp Ala Glu Arg Gly Lys Leu Lys Arg Arg Ala 225 230 235 240 Ile Ala Thr Pro Gly Met Gln Ile Arg Gly Phe Val Tyr Phe Val Glu 245 250 255 Thr Leu Ala Arg Arg Ile Cys Glu Lys Leu Glu Gln Ser Gly Leu Pro 260 265 270 Val Gly Gly Asn Glu Lys Lys Ala Lys Leu Ala Asn Val Val Arg Lys 275 280 285 Met Met Thr Asn Ser Gln Asp Thr Glu Leu Ser Phe Thr Ile Thr Gly 290 295 300 Asp Asn Thr Lys Trp Asn Glu Asn Gln Asn Pro Arg Met Phe Leu Ala 305 310 315 320 Met Ile Thr Tyr Ile Thr Arg Asn Gln Pro Glu Trp Phe Arg Asn Val 325 330 335 Leu Ser Ile Ala Pro Ile Met Phe Ser Asn Lys Met Ala Arg Leu Gly 340 345 350 Lys Gly Tyr Met Phe Glu Ser Lys Ser Met Lys Leu Arg Thr Gln Ile 355 360 365 Pro Ala Glu Met Leu Ala Ser Ile Asp Leu Lys Tyr Phe Asn Asp Ser 370 375 380 Thr Lys Lys Lys Ile Glu Lys Ile Arg Pro Leu Leu Val Asp Gly Thr 385 390 395 400 Ala Ser Leu Ser Pro Gly Met Met Met Gly Met Phe Asn Met Leu Ser 405 410 415 Thr Val Leu Gly Val Ser Ile Leu Asn Leu Gly Gln Arg Lys Tyr Thr 420 425 430 Lys Thr Thr Tyr Trp Trp Asp Gly Leu Gln Ser Ser Asp Asp Phe Ala 435 440 445 Leu Ile Val Asn Ala Pro Asn His Glu Gly Ile Gln Ala Gly Val Asp 450 455 460 Arg Phe Tyr Arg Thr Cys Lys Leu Val Gly Ile Asn Met Ser Lys Lys 465 470 475 480 Lys Ser Tyr Ile Asn Arg Thr Gly Thr Phe Glu Phe Thr Ser Phe Phe 485 490 495 Tyr Arg Tyr Gly Phe Val Ala Asn Phe Ser Met Glu Leu Pro Ser Phe 500 505 510 Gly Val Ser Gly Ile Asn Glu Ser Ala Asp Met Ser Ile Gly Met Thr 515 520 525 Val Ile Lys Asn Asn Met Ile Asn Asn Asp Leu Gly Pro Ala Thr Ala 530 535 540 Gln Met Ala Leu Gln Leu Phe Ile Lys Asp Tyr Arg Tyr Thr Tyr Arg 545 550 555 560 Cys Gln Arg Gly Asp Thr Gln Ile Gln Thr Arg Arg Ser Phe Glu Leu 565 570 575 Lys Lys Leu Trp Glu Gln Thr Arg Ser Lys Thr Gly Leu Leu Val Ser 580 585 590 Asp Gly Gly Pro Asn Leu Tyr Asn Ile Arg Asn Leu His Ile Pro Glu 595 600 605 Val Cys Leu Lys Trp Glu Leu Met Asp Glu Asp Tyr Lys Gly Arg Leu 610 615 620 Cys Asn Pro Leu Asn Pro Phe Val Ser His Lys Glu Ile Glu Ser Val 625 630 635 640 Asn Ser Ala Val Val Met Pro Ala His Gly Pro Ala Lys Ser Met Glu 645 650 655 Tyr Asp Ala Val Ala Thr Thr His Ser Trp Ile Pro Lys Arg Asn Arg 660 665 670 Ser Ile Leu Asn Thr Ser Gln Arg Gly Ile Leu Glu Asp Glu Gln Met 675 680 685 Tyr Gln Lys Cys Cys Asn Leu Phe Glu Lys Phe Phe Pro Ser Ser Ser 690 695 700 Tyr Arg Xaa Pro Val Gly Ile Ser Ser Met Val Glu Ala Met Val Ser 705 710 715 720 Arg Ala Arg Ile Asp Ala Arg Ile Asp Phe Glu Ser Gly Arg Ile Lys 725 730 735 Lys Asp Glu Phe Ala Glu Ile Met Lys Ile Cys Ser Thr Ile Glu Glu 740 745 750 Leu Arg Arg Gln Lys 755 108 2271 DNA Equine influenza virus H3N8 108 atggatgtca atccgactct actcttctta aaggtgccag cgcaaaatgc tataagcaca 60 acattccctt atactggaga tcctccctac agtcatggaa cagggacagg atacaccatg 120 gatactgtca acagaacaca tcaatactca gaaaagggga aatggacaac aaacactgag 180 attggagcac cacaacttaa tccaatcgat ggaccgcttc ctgaagacaa tgaaccaagt 240 gggtacgccc aaacagattg tgtattggaa gcaatggctt tccttgaaga atcccatccc 300 ggaatctttg aaaattcgtg tcttgaaaca atggaggtgg ttcagcagac aagagtggac 360 aaactaacac aaggccgaca aacttacgat tggaccttga ataggaatca acctgccgca 420 acagcacttg ctaatacaat tgaagtgttc agatcaaatg atctgacttc cagtgagtca 480 gggagattaa tggacttcct caaagatgtc atggagtcca tgaacaagga agaaatggaa 540 ataacaacac acttccaacg gaagagaaga gtaagagaca acatgacaaa gagaatggtg 600 acacagagaa ccatagggaa gaaaaaacaa cgattaaaca gaaagagcta tctgatcagg 660 gcattaacct taaacacaat gaccaaggac gctgagagag ggaaattgaa acgacgagca 720 attgcaaccc caggaatgca gataagaggg tttgtatatt ttgttgaaac attagcccga 780 agaatatgtg aaaagcttga acaatcagga ttgccagttg gcggtaatga gaaaaaggcc 840 aaactggcta atgtcgtcag aaaaatgatg actaattccc aagacactga actctccttc 900 accatcactg gggacaatac caaatggaat gaaaatcaga acccacgcat gttcctggca 960 atgatcacat acataactag aaaccagcca gaatggttca gaaatgttct aagcattgca 1020 ccgattatgt tctcaaataa aatggcaaga ctggggaaag gatatatgtt tgaaagcaaa 1080 agtatgaaat tgagaactca aataccagca gaaatgctcg caagcattga tctgaaatat 1140 ttcaatgatt caacaaaaaa gaaaattgag aagatacgac cacttctggt cgatgggact 1200 gcttcactga gtcctggcat gatgatggga atgttcaaca tgttgagcac tgtactaggt 1260 gtatccatat taaacctggg ccagaggaaa tacacaaaga ccacatactg gtgggatggt 1320 ctgcaatcat ccgatgattt tgctttgata gtgaatgcgc ctaatcatga aggaatacag 1380 gctggagtag acagattcta tagaacttgc aaactggtcg ggatcaacat gagcaaaaag 1440 aagtcctaca taaatagaac cggcacattc gaattcacaa gctttttcta ccggtatggt 1500 tttgtcgcca atttcagcat ggagctaccc agttttgggg tttccgggat aaatgaatct 1560 gcagacatga gcattggaat gacagttatc aaaaacaaca tgataaataa tgatctcggt 1620 cccgccacgg cacaaatggc actccaactc ttcattaagg attatcggta cacataccgg 1680 tgtcaaagag gcgataccca gatacaaacc agaagatcct ttgagttgaa gaaactgtgg 1740 gaacagactc gatcaaagac tggtctactg gtatcagatg ggggtccaaa cctatacaac 1800 atcagaaacc tacacatccc ggaagtctgt ttgaaatggg agctgatgga tgaagattat 1860 aaagggaggc tatgtaatcc attgaatcct ttcgttagcc acaaagaaat tgaatcagtg 1920 aacagtgcag tagtaatgcc tgcgcatggc cctgccaaaa gcatggagta tgatgctgtt 1980 gcaacaacac actcttggat ccccaagagg aaccggtcca tattgaacac aagtcaaagg 2040 ggaatactcg aagatgagca gatgtatcag aaatgctgca acctgtttga aaaattcttc 2100 cccagcagct catacagaar accagtcgga atttctagta tggttgaggc catggtgtcc 2160 agggcccgca ttgatgcacg aattgacttc gaatctggac ggataaagaa ggatgagttc 2220 gctgagatca tgaagatctg ttccaccatt gaagagctca gacggcaaaa a 2271 

What is claimed is:
 1. An isolated equine influenza nucleic acid molecule selected from the group consisting of: a. an insolated nucleic acid molecule that encodes a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO:69, SEQ ID NO:92, and SEQ ID NO:107; and b. an isolated nucleic acid molecule fully complementary to a nucleic acid molecule of (a); wherein said nucleic acid molecule of (a) or (b) is not an entire equine influenza virus genome.
 2. The nucleic acid molecule of claim 1, wherein said nucleic acid molecule comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:106 and SEQ ID NO:108 and a nucleic acid molecule comprising a nucleic acid sequence which is fully complementary to any of said nucleic acid sequences.
 3. A nucleic acid molecule of claim 1, wherein said nucleic acid molecule encodes a protein.
 4. A nucleic acid molecule of claim 1, wherein said nucleic acid molecule encodes a protein selected from the group consisting of Pei_(ca1)PB1-C₃₉₆, and Pei_(ca1)PB1₇₅₇. 